Antioxidant producing bacterium and uses thereof

ABSTRACT

Bacterial strains are provided that can be isolated from the microflora of lowbush blueberry ( Vaccinium angustifolium ), and that are capable of increasing the antioxidant content of their growth medium. The bacteria can be used, for example, to increase the antioxidant content of various foodstuffs, as probiotics or as additives to animal feed. Antioxidant-enriched compositions produced by fermentation processes utilising the bacteria are also provided. The antioxidant-enriched compositions can be used in the preparation of cosmetics and nutritional supplements. The antioxidant-enriched compositions also have therapeutic applications.

FIELD OF THE INVENTION

The present invention pertains to the field of bacterial fermentation,in particular to an antioxidant producing bacterium and the use thereofin fermentation to produce antioxidants.

BACKGROUND OF THE INVENTION

Naturally occurring plant derived compounds are believed to afford somehealth benefits due in part to their antioxidant properties. Theseantioxidant activities may be important in preventing, treating orameliorating many diseases, for example, cancer, neurodegenerativediseases such as Parkinson's and Alzheimer's, cardiovascular disease,and inflammation, as well as various conditions related to aging.Indeed, there is growing evidence that these compounds may be beneficialas chemopreventative, anti-inflammatory, immunomodulatory orcardioprotective agents. Accordingly, there is extensive researchdirected at identifying, extracting, purifying and developing plantderived antioxidant compositions for use as cosmetics, dietarysupplements/nutraceuticals, food additives or pharmaceuticals.

Phenolic compounds, including their subcategory, flavonoids, arena groupof compounds that possess antioxidant properties, which are present inall plants and have been studied extensively in cereals, legumes, nuts,olive oil, vegetables, fruits, tea and red wine. There is considerableinterest in these naturally occurring antioxidants and in methods ofextracting these compounds from various plants and plant products. Forexample, U.S. Pat. No. 6,620,452 describes a process for extractingplant phenolics from a fruit or vegetable, and steps to provide a liquidor solid concentrate thereof. U.S. Pat. No. 5,932,623 describes aprocess for obtaining extracts from unripe fruits and purports toidentify the polyphenol products that are present in the extract andU.S. Pat. No. 5,994,413 describes a mixture containing polyphenolproducts extracted from unripe Suits.

Antioxidants, including phenolics, have been identified in, andextracted from, various berries. For example, U.S. Pat. No. 6,676,978describes a method for isolating a mixture of anthocyanins,bioflavonoids and phenolics from an edible berry using adsorbent resins.The use of compositions comprising these compounds to provideantioxidant and anti-inflammatory activities to a mammal is alsodescribed. U.S. Pat. No. 6,312,745 describes a process for dehydratingberries while maintaining their antioxidant compounds/activities. U.S.Patent Application No. 2003/0031734 is directed to blueberry extractswith anti-oxidant and anti-cancer properties and their use to inhibittumour cell growth and oxidative activity in an animal.

Many plant derived antioxidants have been proposed for use as dietarysupplements. For example, U.S. Patent Application No. 2002/0068102describes a dietary supplement comprising natural juices and its use toreduce cellular damage by scavenging free radicals within the humanbody. The natural juices can be derived from, for example, natural grapeconcentrate, a natural blueberry juice concentrate and/or other naturaljuice concentrates. U.S. Patent Application No. 2003/0008048 describes adietary nutritional supplement that may comprise blueberry, for helpingthe body resist the effects of the aging process and U.S. PatentApplication No. 2003/003120 is directed to a phenolic fraction obtainedfrom fruit and its use as a cosmetic, dietary, or nutraceuticalpreparation. Nutritional supplements are also described in U.S. PatentApplication No. 2002168429. This patent application describes a methodof producing reconstituted vegetable or fruit products that can then beused to prepare a dietary supplement. Fruit juices contemplated by thisapplication include blueberry and cranberry juices, which are expressedfrom the berries and subsequently concentrated.

Fermentation can also be employed to modulate the antioxidant content ofa plant derived products. For example, U.S. Pat. No. 5,498,412 describesa method for producing a natural antioxidant composition made from aplurality of fermented and milled materials of edible grains and pulses.In addition, wine fermentation has been reported to bring about multiplechemical modifications with respect to phenolic antioxidant profiles(Mazza, G., et al., (1999) J. Agric. Food Chem., 47, 4009-4017; Talcott,S. T. & J. H. Lee (2002) J. Agric. Food Chem., 50, 3186-3192).

Thus, the antioxidant content of fruits and vegetables variesconsiderably not only with species and growth conditions, but also witha number of other factors, including processing, fermentation, exposureto various temperatures, irradiation and pathogenic infection.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an antioxidantproducing bacterium and uses thereof. In accordance with an aspect ofthe present invention, there is provided a bacterial strain having allthe identifying characteristics of the bacterium deposited underAccession Number 160103.

In accordance with another aspect of the present invention, there isprovided a bacterial strain having a 16S rRNA gene comprising anucleotide sequence that is at least about 97% identical to the sequenceas set forth in SEQ ID NO:1.

In accordance with another aspect of the present invention, there isprovided a process for producing an antioxidant-enriched fruit extractcomprising: fermenting a fruit extract with a bacterial strain of theinvention.

In accordance with another aspect of the present invention, there isprovided a process for producing an antioxidant-enriched fruit extract,said method comprising: providing a sterile medium comprising a fruitextract, inoculating said sterile medium with a bacterial strain havingall the identifying characteristics of the bacterium deposited underAccession Number 160103 to provide a bacterial culture; fermenting saidbacterial culture to provide an antioxidant-enriched fruit extract, andrecovering said antioxidant-enriched fruit extract.

In accordance with another aspect, there is provided anantioxidant-enriched fruit extract produced by a process of theinvention.

In accordance with another aspect of the present invention, there isprovided an antioxidant composition comprising a carrier or diluent andan antioxidant-enriched fruit extract produced by a process of theinvention.

In accordance with another aspect, there is provided a use of anantioxidant-enriched fruit extract of the invention in the preparationof a cosmetic composition.

In accordance with another aspect, there is provided a use of anantioxidant-enriched fruit extract of the invention in the preparationof a pharmaceutical composition.

In accordance with another aspect, there is provided a use of anantioxidant-enriched fruit extract of the invention in the preparationof a nutraceutical, functional food, beverage or dietary supplement.

In accordance with another aspect, there is provided a use of anantioxidant-enriched fruit extract of the invention to deliverantioxidants to a mammal in need thereof.

In accordance with another aspect of the invention, there is provided acosmetic composition produced by a process comprising the steps of:fermenting a fruit extract with a bacterial strain of the invention toprovide an antioxidant-enriched fruit extract; and combining saidantioxidant-enriched fruit extract with a cosmetically acceptablecarrier or diluent.

In accordance with another aspect of the invention, there is provided apharmaceutical composition produced by a process comprising the stepsof: fermenting a fruit extract with a bacterial strain of the inventionto provide an antioxidant-enriched fruit extract; and combining saidantioxidant-enriched fruit extract with a pharmaceutically acceptablecarrier or diluent.

In accordance with another aspect of the present invention, there isprovided a cosmetic composition comprising an antioxidant-enriched fruitextract of the invention and a cosmetically acceptable carrier ordiluent.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical composition comprising an antioxidant-enrichedfruit extract of the invention and a pharmaceutically acceptable carrieror diluent.

In accordance with another aspect, there is provided a use of apharmaceutical composition of the invention as an antioxidant in amammal in need thereof.

In accordance with another aspect, there is provided a use of apharmaceutical composition of the invention as an immunomodulator in amammal in need thereof.

In accordance with another aspect, there is provided a use of apharmaceutical composition of the invention to stimulate TNF-αproduction in a mammal in need thereof.

In accordance with another aspect, there is provided a use ofα-bacterial strain of the invention in a fermentation process.

In accordance with another aspect, there is provided a use of abacterial strain of the invention as a food additive.

In accordance with another aspect, there is provided a use of abacterial strain of the invention to increase the antioxidant content ofa food product.

In accordance with another aspect, there is provided a use of abacterial strain of the invention to metabolise toxic phenolic compoundsin a sample.

In accordance with another aspect, there is provided a compositioncomprising a bacterial strain of the invention and a suitable medium orstabiliser.

In accordance with another aspect, there is provided a kit comprising abacterial strain of the invention and optionally one or more growthmedium ingredient for propagation of the bacterial strain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a 1500 nucleotide sequence of the 16S rRNA gene of thebacterium deposited under with the International Depository Authority ofCanada under Accession Number 160103 [SEQ ID NO: 1].

FIG. 2 depicts the phylogenetic analysis based on the partial 16S rRNAfragment for the bacterium (Accession No. 160103).

FIG. 3 depicts the total phenolic content in GAE (mg of gallic acidequivalent/100 g of fresh weight), during fermentation of blueberry bythe bacterium (Accession No. 160103). Error bars indicate standarddeviation for each day of fermentation.

FIG. 4 depicts the difference in radical scavenging activity (RSA)between non-fermented and fermented blueberry juice samples after 3 daysof fermentation with (A) same total phenolic concentration and (B)different total phenolic concentration. Increase in RSA over time for(C) non-fermented blueberries and (D) fermented blueberries.

FIG. 5 depicts the phenolic profiles derived using (A) capillaryelectrophoresis for separation, and diode array detection at 280 nm ofunfermented (positive signal) and fermented (negative signal) blueberrysamples. (B) The UV spectrum of the main peak obtained from fermentedblueberry sample. HPLC derived phenolic profiles are depicted for (C)unfermented blueberry and (D) fermented blueberry samples. Peaks ofinterest are: (1) gallic acid; (2) unknown phenolic acid; (3)chlorogenic acid.

FIG. 6 depicts (A) the total phenolic content in GAE (mg of gallic acidequivalent/100 g of fresh weight) during fermentation of cranberry juiceby the bacterium (Accession No. 160103) [Error bars indicate standarddeviation for each day of fermentation.] and (B) the total phenoliccontent in GAE during fermentation of grape juice by the bacterium(Accession No. 160103).

FIG. 7 depicts pH variation during cranberry juice fermentation by thebacterium (Accession No. 160103).

FIG. 8 depicts the total phenolic content in GAE (mg of gallic acidequivalent/100 g of fresh weight) during fermentation of cranberry juice(A) by the bacterium (Accession No. 160103) under anaerobic conditionsand (B) by a mixture of the bacterium (Accession No. 160103) and wineyeast. Error bars indicate standard deviation for each day offermentation.

FIG. 9 depicts the antiradical power of unfermented and fermentedcranberry juice by the bacterium (Accession No. 160103).

FIG. 10 depicts the inhibition of nitric oxide production (NO) fromLPS/IFN-γ-activated macrophages by (A) cranberry juice, (B) blueberryjuice and (C) phenolic compounds.

FIG. 11 depicts the relative inhibitory effect (%) of (A) cranberryjuice, blueberry juice and phenolic compounds on nitric oxide production(NO) from LPS/IFN-γ-activated macrophages. Inhibitory effect achievedwith (B) 125 μM GAE and (C) 250 μM GAE.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a previously unidentified bacterialspecies that can be isolated from the microflora of lowbush blueberry(Vaccinium angustifolium) and which is capable of increasing theantioxidant content of its growth medium. The bacteria of the inventioncan be used, for example, to increase the antioxidant content of variousfoodstuffs, as a probiotic or as an additive to animal feed. Thebacteria can also be used in fermentation processes to produceantioxidant-enriched fruit extracts, and in particularantioxidant-enriched berry extracts. The present invention thus furtherprovides for antioxidant-enriched compositions produced by fermentingfruit extracts with a bacterium of the invention. Such compositions haveapplication in the preparation of cosmetics, as dietary or nutritionalsupplements and food additives. The antioxidant-enriched compositionsare also useful as therapeutics in the treatment of a disease, disorderor condition in a mammal associated with free radical damage or theformation of reactive oxygen species (ROS). The compositions of theinvention have been demonstrated to have immunomodulatory properties andthus also have applications as therapeutics in the treatment of diseasesand disorders related to the immune system.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

The term “sequence identity,” as used herein, means that twopolynucleotide sequences, a candidate sequence and a reference sequence,are identical (i.e. on a nucleotide-by-nucleotide basis) over the lengthof the candidate sequence. In comparing a candidate sequence to areference sequence, the candidate sequence may comprise additions ordeletions (i.e. gaps) of 20 percent or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences fordetermining sequence identity may be conducted using the local alignmentalgorithm of Smith and Waterman (Adv. Appl. Math. (1981) 2:482), thealignment algorithm of Needleman and Wunsch (J. Mol. Biol. (1970)48:443), the search for similarity method of Pearson and Lipman (Proc.Natl. Acad. Sci. (U.S.A.) (1988) 85:2444), using computerisedimplementations of these algorithms (such as GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 573 Science Dr., Madison, Wis.), using publiclyavailable computer software such as ALIGN or Megalign (DNASTAR), or byinspection.

The term “percent (%) sequence identity,” as used herein with respect toa reference sequence is defined as the percentage of nucleotide residuesin a candidate sequence that are identical to the residues in thereference polynucleotide sequence after optimal alignment of thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

The term “specifically hybridise,” as used herein, refers to the abilityof a nucleic acid sequence to bind detectably and specifically to asecond nucleic acid sequence. A polynucleotide selectively hybridises toa target nucleic acid sequence under hybridisation and wash conditionsthat minimise appreciable amounts of detectable binding to non-specificnucleic acid sequences. High stringency conditions can be used toachieve specific hybridisation conditions as known in the art.Typically, hybridisation and washing conditions are performed at highstringency according to conventional hybridisation procedures. Washingconditions are typically 1-3×SSC, 0.1-1% SDS, 50-70° C. with a change ofwash solution after about 5-30 minutes.

The term “fruit extracts,” as used herein, refers to a preparationderived from a fruit or one or more part thereof, including juice, peel,rind, pith, pips and flesh. The preparation can be derived from thefruit or fruit part by a variety of techniques, including for example,maceration, squeezing, pressing, expressing, distillation, steeping,soaking, infusion, filtration or concentration of the fruit or fruitpart. Combinations of these techniques can also be used.

“Substantially pure’ or “isolated,” as used herein with reference to acompound found in a fruit extract, indicates that the compound is thepredominant species present in a composition (i.e. on a molar basis itis more abundant than any other individual compound in the composition).Typically, substantially pure that the compound constitutes at leastabout 20% of all macromolecular species present in the composition. Inone embodiment, a substantially pure compound refers to a compound thatconstitutes more than about 30% of all macromolecular species present ina composition. In another embodiment, a substantially pure compoundrefers to a compound that constitutes more than about 40% of allmacromolecular species present in a composition. In other embodiments, asubstantially pure compound refers to a compound that constitutes morethan about 50%, more than about 60% and more than about 70% of allmacromolecular species present in a composition.

As used herein, the term “about” refers to a +/−10% variation from thenominal value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

Bacteria of the Present Invention

A representative bacterium of the present invention was isolated fromthe microflora of lowbush blueberry (Vaccinium angustifolium) anddeposited with the International Depository Authority of Canada on Jan.16, 2003 under Accession Number 160103. Analysis of the biochemicalprofile and partial sequence of the 16S rRNA gene of the bacterium asdescribed herein indicates that it belongs to the familyEnterobacteriaceae.

The bacterium deposited under Accession Number 160103 is characterisedin that it is a gram negative, catalase positive, facultativelyanaerobic coccobacillus. The bacterium has a fermentative metabolism andcan ferment a number of sugars, including D-glucose, D-fructose,D-mannose, arbutin, esculin, salicin, saccharose and D-raffinose. Undercertain conditions, the bacterium can also ferment mannitol, lactose andtrehalose as described herein in Example I. The bacterium producesacetoin, hydrolyses hippurate and produces a number of enzymesincluding, pyrrolidonyl arylamidase, α-galactosidase, β-galactosidase,alkaline phosphatase and leucine arylamidase.

The bacterium deposited under Accession Number 160103 can further becharacterised as having a 16S rRNA gene sequence that comprises thenucleotide sequence as set forth in SEQ ID NO:1.

As is known in the art, bacterial species are often represented by anumber of different strains that share the same characteristics. Thepresent invention, therefore, encompasses strain variants of thebacterium deposited under Accession Number 160103. In accordance withthe present invention, these strains possess the same biochemicalcharacteristics as outlined above for the bacterium deposited underAccession Number 160103.

The biochemical characteristics of the bacteria encompassed by thepresent invention can readily be determined using standard techniquesknown in the art. For example, the bacterium can be identified asgram-negative by the fact it does not retain crystal violet stain in thepresence of alcohol or acetone. The fermentative abilities of thebacterium can be determined, for example, by using one of a variety ofkits available commercially for this purpose (for example, the API andVITEK kits from BioMérieux, Marcy-l'Éttoile, France).

As indicated above, the bacterium deposited under Accession Number160103 has a 16S rRNA gene sequence that comprises the nucleotidesequence as set forth in SEQ ID NO:1. It is well known in the art,however, that bacteria of the same species need not share 100% sequenceidentity in their 16S rRNA gene sequences and it is generally acceptedthat a 3% variation between the 16S rRNA gene sequences of two bacteriais the point at which two strains may be considered to be separatespecies (see, for example, Vandamme, et al., (1996) Microbiol. Reviews60:407-48; Kolbert & Persing, (1999) Curr. Microbiol., 2:299-305). Thus,a species is defined by at least 97% sequence identity in the 16S rRNAgene sequence. Bacteria considered to be within the scope of the presentinvention, therefore, are characterised in that their 16S rRNA genecomprises a sequence that is at least 97% identical to the sequence asset forth in SEQ ID NO:1. In one embodiment, the bacteria arecharacterised in that their 16S rRNA gene comprises a sequence that isat least 97.5% identical to the sequence as set forth in SEQ ID NO:1. Inanother embodiment, the bacteria are characterised in that their 16SrRNA gene comprises a sequence that is at least 98% identical to thesequence as set forth in SEQ ID NO:1. In a further embodiment, thebacteria are characterised in that their 16S rRNA gene comprises asequence that is at least 98.2% identical to the sequence as set forthin SEQ ID NO:1. In still another embodiment, the bacteria arecharacterised in that their 16S rRNA gene comprises a sequence that isat least 98.5% identical to the sequence as set forth in SEQ ID NO:1. Inother embodiments, the bacteria are characterised in that their 16S rRNAgene comprises a sequence that is at least 98.8%, at least 99% and atleast 99.5% identical to the sequence as set forth in SEQ ID NO:1.

In an alternate embodiment, the bacteria are characterised in that thesequence of the 16S rRNA gene comprises at least 100 consecutivenucleotides of the sequence as set forth in SEQ ID NO:1, for example, atleast 250 consecutive nucleotides of the sequence as set forth in SEQ IDNO:1. In other embodiments, the 16S rRNA gene of the bacteria comprisesat least 500, 750 or 1000 consecutive nucleotides of the sequence as setforth in SEQ ID NO:1.

Sequencing of the 16S rRNA gene of the bacterium of interest can bereadily conducted using DNA isolation and sequencing techniques known inthe art [see, for example, Ausubel et al., Current Protocols inMolecular Biology, J. Wiley & Sons, NY] or using commercially availablekits such as the MicroSeq™ 16S rRNA Gene Kit and software, availablefrom Applied Biosystems. Comparison of the identified sequence with thatset forth in SEQ ID NO:1 can be conducted using standard techniquesincluding, for example, the use of publicly available software, such asBLAST (available from the NCBI website) and CLUSTALW (available from theEMBL-EBI website).

The present invention thus also provides for methods of identifyingbacterial strains of the same species as the bacterium deposited underAccession Number 160103, which are capable of increasing the phenolicantioxidant content of a growth medium. The methods employ nucleic acidprobes comprising the sequence set forth in SEQ ID NO:1, or a fragmentthereof, to screen DNA derived from other bacterial strains to identifythose whose 16S rRNA gene DNA specifically hybridises to the nucleicacid probe. Methods of screening bacterial DNA samples for sequencesthat hybridise to a known probe are standard in the art [see, forexample, Ausubel et al., Current Protocols in Molecular Biology, J.Wiley & Sons, NY]. Bacteria that are identified as having DNA sequencesthat specifically hybridise to the probe can then be analysed further bysequence analysis.

If necessary, PCR primers can be designed based on the sequence setforth in SEQ ID NO:1 that would allow amplification of target DNA fromthe bacterium under investigation in order to facilitate hybridisationscreening and/or sequence analysis. Design of appropriate primers fromthe sequence set forth in SEQ ID NO:1 is considered to be within theordinary skills of a worker in the art. Alternatively, primers designedto amplify the hypervariable portion of the 16S rRNA gene can be basedon the sequences that flank this portion of the gene ad which areconserved in most Eubacteria. Kits for the isolation and sequencing ofbacterial 16S rRNA genes are commercially available (for example, theMicroSeq® Microbial Identification System from Applied Biosystems,Foster City, Calif.). Bacteria having a 16S rRNA gene sequence thatcomprises a sequence at least 97% identical to SEQ ID NO:1 are selectedand their biochemical profiles are assessed for similarity to that ofthe bacterium deposited under Accession No. 160103.

Alternatively, the methods of identifying the bacteria of the inventioncan comprise first isolating a bacterium from lowbush blueberries andsubsequently determining the biochemical profile of the isolatedbacterium as described above. Bacteria having a biochemical profile thatmatches that of the bacterium deposited under Accession No. 160103 areselected. Analysis of the 16S rRNA gene sequence is then conducted andthose bacteria having a 16S rRNA gene sequence that comprises a sequenceat least 97% identical to the sequence as set forth in SEQ ID NO:1 areselected as bacteria of the invention.

Bacterial strains selected by the above methods can be further testedfor their ability to increase the antioxidant content of a growth mediumusing techniques known in the art such as those described herein.

Isolation and Propagation

The bacteria of the present invention can be isolated from lowbushblueberry microflora by the methods described herein.

For example, in one embodiment of the present invention, the bacteriumis isolated from the surface of lowbush blueberries by inoculation of atryptic soy broth with whole lowbush blueberry fruits, followed bygrowth at 25° C. for 36 hrs, and subsequent selection on tryptic soyagar by serial dilution. Each bacterial isolate can then be tested forantioxidant production during fermentation, for other biochemicalproperties as described above, or by sequence analysis in order todetermine whether it is a bacterium of the invention.

The bacteria of the invention can be maintained and propagated on avariety of different media using standard culture techniques. Examplesof suitable media include; but are not limited to, tryptic soy broth oragar, Simmons citrate agar, MRS agar, Voges-Proskauer agar and potatodextrose agar. In one embodiment of the present invention, the bacteriumis maintained and/or propagated on tryptic soy broth or agar.

For propagation, the bacteria of the invention can be grown at atemperature between about 8° C. and about 36° C., however, the growthrate at temperatures between 10° C. and 8° C. is typically diminishedcompared to that at higher temperatures. In one embodiment of theinvention, therefore, the bacteria are propagated between about 10° C.and about 36° C. In another embodiment, the bacteria are propagatedbetween about 20° C. and about 25° C.

Genetic Engineering

The present invention further contemplates genetic engineering of thebacterium in order to improve certain desirable traits and/or todecrease or eliminate less desirable traits. For example, the bacteriumcould be engineered to enhance the production of antioxidant compounds.Methods of genetically engineering bacteria are well known in the art.

Antioxidant-Enriched Compositions

The present invention further provides for antioxidant-enrichedcompositions comprising fruit extracts that have been fermented with abacterium of the invention to enrich their antioxidant content. In oneembodiment of the invention, the fermented fruit extract is enriched inphenolic antioxidants,

The fermented fruit extracts can be partially or substantially purifiedif desired using standard techniques in order to concentrate theantioxidant components further or to isolate one or more antioxidantcompounds therefrom. Thus, the compositions can comprise fermented fruitextracts that have not undergone further processing, or they cancomprise concentrates or solids derived from the fermented fruitextracts, partially purified fermented fruit extracts, or partially orsubstantially purified compounds derived from the fermented fruitextracts.

Production of Fermented Fruit Extracts

The present invention provides for a process of bacterial fermentationto provide antioxidant-enriched Suit extracts.

To produce antioxidant-enriched fruit extracts by fermentation with abacterium of the invention, an appropriate medium is first selected.Typically, the medium contains at least 10% fruit extract together withsufficient amounts and proportions of ions to support bacterial growth.To maximise the amount of antioxidants produced by the fermentationprocess, higher proportions of fruit extracts can be incorporated intothe growth medium. For example, media containing at least 20% fruitextract, at least 30% fruit extract, at least 40% fruit extract, atleast 50% fruit extract, at least 60% fruit extract and at least 70%fruit extract can be used. One skilled in the art will understand thatmedia may be formulated that contain up to 100% fruit extract providedthat sufficient amounts and proportions of ions are incorporated tosupport bacterial growth. One example of an appropriate medium is ablended medium containing about 50% fruit and about 50% minimal media(i.e. water containing sufficient amounts and proportions of ions tosupport bacterial growth).

The pH of the medium is adjusted to between about 5.0 and about 3.3using standard techniques, in order to support the growth of thebacterium.

Fermentation of a medium containing grape or berry extracts by thebacterium deposited under Accession No. 160103 has been shown to resultin an increase in the total amount of antioxidants in the medium. It iscontemplated, however, that the medium can be prepared using extractsderived from one, or a mixture, of a variety of fruits. For example, themedium can be prepared from grapes or from berries including, but notlimited to, blueberries, cranberries, lingonberries, blackcurrants,chokecherries, chokeberries, raspberries, blackberries, elderberries andSaskatoon berries, or various combinations thereof. In one embodiment ofthe present invention, the fruit used in the medium is one or more of:grapes, blueberries, cranberries, elderberries, chokecherries,blackcurrants, and Saskatoon berries. In another embodiment of thepresent invention, the fruit used in the medium is one or more berryfrom the genus Vaccinium (including blueberries, cranberries andlingonberries). The amount of antioxidant-enrichment that is achievedfrom the fermentation will vary depending on the fruit selected and canbe determined using standard procedures such as those described hereinand in the literature.

As is known in the art, many commercially grown fruits are treated withchemicals to prevent the growth of microbial contaminants, which couldinterfere with the ability of the bacteria of the invention to fermentmedia comprising these treated fruits. Thus, in one embodiment of theinvention, the fruit utilised in the preparation if the medium isorganic. In another embodiment, the fruit is a wild fruit.

The medium can be readily prepared, for example, by blending anappropriate amount of one or more fruit with water or a minimal mediumand sterilising the resultant solution. When water is used, salts andions can be added as required. If desired, other components that promotethe growth of the bacterium, such as amino acids or carbohydrates, canbe incorporated into the medium.

The one or more fruit used to prepare the medium can be fresh, frozen,tinned or dried and can be a whole fruit, or a pulp, paste, puree,juice, juice concentrate or solid or a fruit nectar. Alternatively, apowdered form of the fruit can be used, in which case it can bereconstituted in water or directly in minimal medium. “Pulp” and “puree”refer to both heat-treated and non heat-treated whole fruit pieces,which have been mechanically transformed into soft mixture orsuspension, whereas a “paste” refers to a pulp or puree that has beenpartially dehydrated.

The pH of the medium can also be adjusted as necessary such that thestarting pH of the medium is above 3.2. As indicated above, a suitablepH for the medium is in the range from about 5.0 and about 3.3. In oneembodiment of the present invention, the starting pH is about 3.7 toabout 5.0. In other embodiments, the starting pH is between about 4.0and about 5.0 and between about 4.5 and about 5.0.

The suspension can be filtered or centrifuged to remove particulatematter prior to sterilisation, if required. If it is required that themedium has a certain phenolic content prior to fermentation, the mediumis sterilised by filtration rather than by heat, as excessive heat candegrade the phenolic compounds. In one embodiment of the presentinvention, the medium is prepared by blending fresh berries with anequal volume of Minimal Broth Davis without dextrose, centrifuging themedium to remove particulate matter and sterilising by filtrationthrough an appropriate filter.

Fermentation is initiated by inoculation of the medium with anappropriate number of bacterial cells. The inoculant can be in the formof a fraction of a starter culture, as a swab comprising cells takenfrom a culture of the bacteria on a solid phase, such as agar, or as afraction of or swab form a frozen culture of the bacteria. When astarter culture is used, the starter culture can employ the same or adifferent medium, such as one of those described above for propagationof the bacteria.

Methods of fermentation are well-known in the art. In accordance withthe present invention, once the medium has been inoculated with thebacterium, the culture is fermented at a temperature between about 8° C.and about 36° C. In one embodiment, the bacterium is fermented at atemperature between about 10° C. and about 30° C. In other embodiments,the bacterium is fermented at a temperature between about 15° C. andabout 25° C. and between about 20° C. and about 24° C.

As the bacterium is facultatively anaerobic, the fermentation can takeplace under aerobic or anaerobic conditions. In one embodiment, thefermentation is conducted under aerobic conditions. In anotherembodiment, it is conducted under anaerobic conditions.

Typically the fermentation is allowed to proceed for between about 1 dayand about 12 days. Maximal amounts of phenolic antioxidants are usuallyobtained after about 2 to about 5 days of fermentation under aerobicconditions and after about 8 days of fermentation under anaerobicconditions. In one embodiment, therefore, the fermentation proceeds forabout 1 day to about 10 days. In another embodiment, the fermentationproceeds for about 1 day to about 7 days. In other embodiments, thefermentation proceeds for about 2 days to about 5 days, for about forabout 3 days to about 4 days and for about 4 days to about 10 days.

In some instances, the pH of the medium may vary during fermentation andthis variation can affect the antioxidant content. Thus, whenapplicable, the pH of the medium can be monitored and adjusted asnecessary during the fermentation using standard techniques.

Assessing the Antioxidant Content of the Fermented Fruit Extracts

After fermentation with the bacterium, the antioxidant content of thefermented medium can be determined using standard analytical methodsknown in the art. Anthocyanin content can be measured, for example, byspectrophotometric methods such as those described by Fuleki and Francis(J. Food Sci. (1968) 33:73-83). Total phenolic content can be assessed,for example, by the Folin-Ciocalteau method (see, for example, Veliogluet al., (1998) J. Agric. Food Chem. 46:4113-4117) or by chromatographytechniques, such as high performance liquid chromatography (HPLC).

In accordance with one embodiment of the present invention, the phenolicantioxidant content of the fermented medium is increased by at least1.5-fold after a 4-day fermentation under aerobic conditions whencompared to unfermented medium. In another embodiment, the phenolicantioxidant content of the fermented medium is increased by at least1.75-fold after a 4-day fermentation with compared to unfermentedmedium. In a further embodiment, the phenolic antioxidant content of thefermented medium is increased by at least 2-fold after a 4-dayfermentation with compared to unfermented medium.

In an alternate embodiment, the phenolic antioxidant content of thefermented medium is increased by at least 1.5-fold after an 8-dayfermentation under aerobic conditions when compared to unfermentedmedium.

Further Processing and/or Purification of the Fermented Fruit Extracts

In accordance with the present invention, the fermented fruit extractsproduced by the above-described process can be further processed orpurified for preparation of the antioxidant-enriched compositions. Thus,for example, the fermented fruit extracts can be further reduced toprovide a solid or liquid concentrate thereof using standard methodsknown in the art. Alternatively, compounds having antioxidant propertiescan be concentrated in, or isolated from, the fermented fruit extractsby standard chemical techniques, for example, by extraction techniques,filtration techniques, chromatographic techniques, such as HPLC, orelectrophoretic techniques, such as capillary electrophoresis.

“Purifying” a fermented fruit extract in the context of the presentinvention indicates that the extract undergoes substantial or partialpurification, and/or fractionation using one or more of a number oftechniques well known in the art, for example, solid-liquid extraction,liquid-liquid extraction, solid-phase extraction (SPE), membranefiltration, ultrafiltration, dialysis, electrophoresis, solventconcentration, centrifugation, ultracentrifugation, liquid or gas phasechromatography (such as size exclusion, affinity, and the like) with orwithout high pressure, lyophilisation, evaporation, precipitation withvarious “carriers” (such as PVPP, carbon, and the like). One skilled inthe art, would appreciate how to use such techniques, in a sequentialfashion, in order to enrich each successive fraction in the activity ofinterest (i.e. antioxidant capacity) by following its activitythroughout the purification procedure.

Solid-liquid extraction means include the use of various solventsincluding supercritical solvents and extractors such as soxhletextractors, vortex shakers, ultrasounds and the like to enhanceextraction, as well as recovery by filtration, centrifugation andrelated methods as described in the literature (see, for example, R. J.P. Cannell, Natural Products Isolation, Humana Press, 1998). Examples ofsolvents that may be used include, but are not limited to, hydrocarbonsolvents, chlorinated solvents, organic esters, organic ethers,alcohols, water, and mixtures thereof. In the case of supercriticalfluid extraction, the invention also covers the use of modifiers such asthose described in V. H. Bright (Supercritical Fluid Technology, ACSSymp. Set. Vol. 488, ch. 22, 1999).

Liquid-liquid extraction means include the use of various mixtures ofsolvents known in the art, including solvents under supercriticalconditions. Typical solvents include those listed above. Theliquid-liquid extraction can be effected manually, or it can besemi-automated or completely automated, and the solvent can be removedor concentrated by standard techniques in the art (see, for example, S.Ahuja, Handbook of Bioseparations, Academic Press, 2000).

Solid-phase extraction (SPE) techniques include the use of cartridges,columns or other devices known in the art. The sorbents that may be usedwith such techniques include, but are not limited to, silica gel (normalphase), reverse-phase silica gel (modified silica gel), ion-exchangeresins, and fluorisil. The invention also includes the use of scavengerresins or other trapping reagents attached to solid supports derivedfrom organic or inorganic macromolecular materials to remove selectivelyactive ingredients or other constituents from the extracts.

Membrane, reverse osmosis and ultrafiltration means include the use ofvarious types of membranes known in the art, as well as the use ofpressure, vacuum, centrifugal force, and/or other means that can beutilised in membrane and ultrafiltration processes (see, for example, S.Ahuja, Handbook of Bioseparations, Academic Press, 2000).

Dialysis means include membranes having a molecular weight cut-offvarying from less than about 0.5 KDa to greater than about 50 KDa. Theinvention also covers the recovery of purified and/or fractionatedextracts from either the dialysate or the retentate by various meansknown in the art including, but not limited to, evaporation, reducedpressure evaporation, distillation, vacuum distillation, andlyophilization.

Various chromatographic means are known in the art and described in theliterature (see, for example, G. Sofer, L. Hagel, Handbook of ProcessChromatography, Academic Press, 1997) and are suitable for use in thepresent invention. Examples include, but are not limited to, regularcolumn chromatography, flash chromatography, high performance liquidchromatography (HPLC), medium pressure liquid chromatography (MPLC),supercritical fluid chromatography (SFC), countercurrent chromatography(CCC), moving bed chromatography, simulated moving bed chromatography,expanded bed chromatography, one- and two dimension thin-layerchromatography (1D- and 2D-TLC), high performance thin-layerchromatography (HPTLC), and centrifugal thin-layer chromatography(centrifugal TLC). With each chromatographic method, examples ofsorbents that may be used include, but are not limited to, silica gel,alumina, fluorisil, cellulose and modified cellulose, various modifiedsilica gels, ion-exchange resins, size exclusion gels and other sorbentsknown in the art (see, for example, T. Hanai, HPLC: A Practical Guide,RSC Press, UK 1999).

Selective precipitation means includes the use of various solvents andsolvent combinations, the use of temperature changes, the addition ofprecipitant and/or modifiers, and/or modification of the pH by additionof base or acid to effect a selective precipitation of activeingredients or other constituents.

The invention also includes the fractionation, partial purification,and/or purification of active ingredients and extracts by steamdistillation, hydrodistillation, or other related methods ofdistillation known in the art (see, for example, L. M. Harwood, C. J.Moody, Experimental Organic Chemistry, Blackwell ScientificPublications, UK, 1989).

The process of purifying the fermented fruit extracts or compoundsderived therefrom also includes the concentration of the extracts bysolvent removal from the original extract, fractionated extract, orpurified extract. Techniques for solvent removal are known to thoseskilled in the art and include, but are not limited to, rotaryevaporation, distillation (normal and reduced pressure), centrifugalvacuum evaporation (speed-vac), and lyophilization.

Testing the Fermented Fruit Extracts

The fermented fruit extracts can be tested for their antioxidantcapacity and/or their immunomodulatory properties using standardtechniques known in the art. Exemplary protocols are provided herein.

1. Antioxidant Capacity

The antioxidant capacity of the fermented fruit extracts can be testedusing a number of standard methods known in the art. For example,antioxidant capacity can be determined by assessment of the radicalscavenging ability, using such techniques as the DPPH(2,2-diphenyl-1-picrylhydrazyl) method, which is described herein. Othersuitable methods include, but are not limited to, the TEAC (troloxequivalent antioxidant capacity) and ORAC (oxygen radical absorbancecapacity) methods.

The TEAC assay measures the ability of a test extract to prevent orquench free radicals formation. This assay employs2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), whichreacts with hydrogen peroxide in the presence of a peroxidase enzyme toform radical cations. The presence of the radical cations can bedetected optically through their effect on6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox).Inhibition of this free radical formation by a test extract is measuredand provides an indication of the antioxidant capacity of the extract.

The ORAC assay uses 2′-azobis-(2′-amidinopropane)-dihydrochloride (AAPH)to generate peroxyl radicals. The radicals are optically measured as thefluorescence quenching and/or destruction of the algal pigmentβ-phycoerythrin. The assay can be automated by using instruments capableof fluorescence lifetime or total fluorescence measurements, thusallowing the free-radical quenching power of test extracts to beanalysed.

A number of other standard assays to determine the ability of thefermented fruit extracts to trap free radicals can be used in order toprovide an indication of the antioxidant capacity of the extracts. Forexample, the total (peroxyl) radical-trapping antioxidant potential(TRAP) assay can be employed. In this assay a lipid sample is contactedwith AAPH in the presence of a test extract. AAPH initiates formation ofperoxyl radicals and an oxygen electrode is used to measure the ratethat the sample resists peroxidation (for example, by measuring the rateof oxygen uptake). This method is generally most effective for measuringthe antioxidant capacity of lipid soluble extracts.

Another example of a suitable assay is the total oxyradical scavagingspecies (TOSC) assay, which quantifies the reactive oxygen speciesscavaging potential of a test extract. The assay utilises thermaldecomposition of ABAP to generate peroxy radicals, which in turngenerate ethylene gas by oxidatively decomposing α-keto-γ-methiobutyricacid. Test extracts that scavenge the reactive oxygen can prevent ordiminish the formation of ethylene, which can be measured by a gaschromatograph. Electron spin resonance (ESR) spectrophotometric assaysbased on Fremy's salt and galvinoxyl radical scavenging can also be used(see, for example, Schwartz, et al., (2001) Eur. Food Res. Technol.212:319-328).

2. Immunomodulatory Properties

The immunomodulatory properties of the fermented fruit extracts can betested using standard methods known in the art. For example, the abilityof the extracts to activate or inhibit cytokine release or nitric oxideproduction in macrophages can be studied in vitro using a suitablemonocyte/macrophage cell line, such as those available from the AmericanType Culture Collection (ATCC) and various commercial sources (forexample, RAW 264.7 and derivatives). Alternatively,monocytes/macrophages can be obtained from mammalian blood samples usingstandard isolation procedures.

Cultures of monocytes/macrophages can be conducted with various amountsof the fermented fruit extracts and incubated for a suitable length oftime. The cells can be stimulated with LPS and/or IFN-γ as necessary.Stimulation can be conducted prior to, at the same time as or aftercontact with the test extract. The level of one or more cytokine (suchas TNF-α, IL-1, IL-1α, IL-2, IL-4, IL-6, IL-8, GM-CSF) in the cellsupernatants after treatment can be measured, for example, using variouscommercially available ELISA kits. Nitric oxide production can bedetermined using methods based on the Griess reaction for measuringnitrite concentration.

In accordance with one embodiment of the present invention, thefermented fruit extracts are capable of inhibiting monocyte/macrophagenitric oxide production. In another embodiment, the fermented fruitextracts are capable of stimulating TNF-α release from monocytes and/ormacrophages.

The fermented fruit extracts can also be tested for cytotoxicity usingstandard techniques. For example, the extracts can be assayed for theirability to inhibit cell growth in vitro. In general, cells of a specifictest cell line are grown to an appropriate density (e.g. approximately1×10⁴) and the test extract is added. After an appropriate incubationtime (for example 48 to 74 hours), cell density is assessed. Methods ofmeasuring cell density are known in the art, for example, cell densitycan be assessed under a light-inverted microscope by measuring thesurface of the culture plate covered by the cell monolayer; or by usingthe resazurin reduction test (see Fields & Lancaster (1993) Am.Biotechnol. Lab. 11:48-50; O'Brien et al., (2000) Eur. J. Biochem.267:5421-5426 and U.S. Pat. No. 5,501,959), the microculture tetrazolium(MTT) assay (Alley, M C et al, Cancer Research 48:589-601, 1988), thesulforhodamine assay (Rubinstein et al., (1990) J. Natl. Cancer Inst.82:113-118) or the neutral red dye test (Kitano et al., (1991) Euro. J.Clin. Investg. 21:53-58; West et al., (1992) J. Investigative Derm.99:95-100).

The fermented fruit extracts can further be tested for theirimmunomodulatory properties in vivo using suitable murine models. Forexample, a mouse xenograft model using an appropriate cancer cell linecan be used to assess the ability of the extracts to modulate cytokineproduction, macrophage infiltration into the tumour, apoptosis and/ortumour growth. Cytokine production and macrophage infiltration can bemeasured by assessing the presence or absence of appropriate cellularmarkers using histochemical or immunohistochemical techniques as isknown in the art. Apoptosis can be measured, for example, using TUNELstaining. Tumour cell growth can be assessed by measurement of thetumour size or weight over a suitable test period. These and otherstandard methods for determining immunomodulatory effects of testcompounds are known in the art (see, for example, Enna, et al., CurrentProtocols in Pharmacology, J. Wiley & Sons, Inc., New York, N.Y.).

Uses

Bacteria

The bacteria of the present invention can be used as food additives orprobiotics in order to increase the antioxidant content of foods or thebioavailability of antioxidants in the gastro-intestinal tract; Forexample, the bacteria may be added to foods, such as yoghurt, cottagecheese, fruit, legume or other vegetable juice and fruit based snacks.Alternatively, the bacteria can be lyophilised and provided in tabletform as a dietary supplement. The bacteria can also be added to animalfeed in order to improve its antioxidant content.

The present invention, therefore, also provides for a process ofincreasing the antioxidant content of a food or beverage productcomprising fermenting the bacteria with one or more ingredient duringthe production of a food or beverage product.

As demonstrated herein, the bacteria of the invention are compatiblewith the yeast Saccharomyces cerevisae, which is used in the productionof foods, such as breads and baked products, and fermented beverages,such as wine. Wine is known to be is a good source of antioxidants andfermentation using Saccharomyces cerevisae in conjunction with thebacteria of the invention can be used to increase the total amount ofantioxidants present in the final product. In one embodiment of thepresent invention, therefore, the bacterium is added to a wine duringfermentation to increase the antioxidant content thereof. In anotherembodiment, the bacterium is used to increase the antioxidant content ofa bread or baked product.

The bacteria may also have utility in the field of decontamination,wherein the bacteria could be used to metabolise toxic phenoliccompounds in a contaminated sample.

Antioxidant-Enriched Compositions

The antioxidant-enriched compositions comprising fermented fruitextracts have a variety of applications. For example, the compositioncan readily be incorporated into nutritional formulations, such asnutraceuticals, functional food and beverage products and dietarysupplements as a source of antioxidants. The present inventioncontemplates that the composition can be added directly to a food orbeverage product or that it can be tabletted for consumption as asupplement. For example, the composition can be incorporated into afruit-based product such as a fruit bar, fruit-flavoured confectionery,jam, jelly, fruit flavoured beverage, and the like. Tablettedsupplements can be provided in the form of tablets, caplets, hard orsoft gelatine capsules, and the like.

The antioxidant-enriched compositions of the invention are also suitablefor use in cosmetic formulations, for example, in skin and hair careformulations, including those formulated to help reduce stress-relatedeffects, in skin rejuvenating formulations, including those formulatedto help reduce wrinkles and/or aging marks, and in cosmetic formulationsintended to help prevent skin damage by UV radiation.

For cosmetic applications, the compositions can be provided as acosmetically suitable formulation, such as a lotion, gel, cream, liquidcream, ointment, oil base, or as a sprayable liquid form. Suchformulations can include a cosmetically acceptable vehicle to act as adiluent, dispersant or carrier for the composition, so as to facilitatetheir distribution when the formulation is applied to the skin.Cosmetically acceptable vehicles other than water can include liquid orsolid emollients, solvents, humectants, thickeners and powders. Varioustypes of skin benefit ingredients can also be optionally included in thecosmetic formulations. Examples of skin benefit ingredients include, butare not limited to, sunscreens, essentially fatty acids, antioxidants,retinoids and tanning agents. The cosmetic formulations can be packagedin a suitable container to suit the viscosity and intended use. Forexample, a lotion or fluid cream can be packaged in a bottle or aroll-ball applicator, a capsule, a propellant-driven aerosol device or acontainer fitted with a pump suitable for finger operation. When thecomposition is a cream, it can simply be stored in a non-deformablebottle or squeeze container, such as a tube or a lidded jar.

It is further contemplated that the compositions can be used asadditives for the preservation of various foodstuffs, as stabilisers inpersonal care formulations, as preservatives in packaging forfoodstuffs, such as cereal boxes and the like, and as preservativesagainst oxidative degradation of plastics.

Pharmaceutical Applications

The present invention provides for the use of the antioxidant-enrichedcompositions to treat, ameliorate or prevent a disease, disorder orcondition in a mammal associated with free radical damage or theformation of reactive oxygen species (ROS). Examples of such diseases,disorders or conditions include cancer, diabetes mellitus,neurodegenerative diseases (such as multiple sclerosis, Parkinson'sdisease, Alzheimer's disease, dementia), arthritis, atherosclerosis,coronary heart diseases, cataracts, cognitive dysfunction, skin photoaging, skin wrinkles, sunburn, melanoma, and degenerative processesassociated with aging.

The present invention further provides for the use of the compositionsas immunomodulators for the treatment of a disease or disorder involvingthe immune system in a mammal. The ability of the compositions tostimulate TNF release also renders them useful in the treatment ofinfectious diseases, cancer and endometriosis.

When used therapeutically, the compositions of the invention can be usedprimarily as a pharmaceutical or drug, or they may be used in thecontext of complementary medicine where the aim is the supplement theeffects of, or decrease the side effects associated with, conventionalmedicine. Thus, the compositions can be used as an adjuvant toconventional drugs, or they can be used to help decrease the sideeffects associated with the drugs, for example, with cancerchemotherapeutics.

For pharmaceutical applications, the compositions are formulated aspharmaceutically acceptable compositions by admixture with aphysiologically acceptable carrier, excipient, binder or diluent.

The pharmaceutical compositions according to the invention can be insolid, semisolid or liquid from and can be adapted for oral, parenteral,rectal, inhalation, or topical administration, and can be provided inunit dosage form. The pharmaceutical composition may be adapted for slowrelease in vivo as known in the art. The pharmaceutical compositions maybe used in conventional form including, but not limited to, solutions,syrups, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, elixirs,injectables, tablets, capsules, suppositories, hydrophobic andhydrophilic creams and lotions. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intrathecal,intramuscular, intrasternal injection or infusion techniques. Otherdrugs may be included in the pharmaceutical composition if desired.

Pharmaceutical compositions intended for oral use may be preparedaccording to methods known in the art and may contain one or more agentssuch as sweetening agents, flavouring agents, colouring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the antioxidant-enrichedcompositions in admixture with non-toxic pharmaceutically acceptableexcipients which are suitable for the manufacture of tablets. Theseexcipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate: granulating and disintegrating agents for example, cornstarch, or alginic acid: binding agents, for example starch, gelatine oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Pharmaceutical compositions for oral use may also be presented as hardgelatine capsules wherein the antioxidant-enriched composition is mixedwith an inert solid diluent, for example, calcium carbonate, calciumphosphate or kaolin, or as soft gelatine capsules wherein the activeingredient is mixed with water or an oil medium, for example peanut oil,liquid paraffin or olive oil.

Aqueous suspensions contain antioxidant-enriched compositions inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example, sodiumcarboxymethylcellulose, methyl cellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample hepta-decaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxy-benzoate, one or more colouringagents, one or more flavouring agents or one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending theantioxidant-enriched compositions in a vegetable oil, for example,arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oilsuch as liquid paraffin. The oily suspensions may contain a thickeningagent, for example beeswax, hard paraffin or cetyl alcohol. Sweeteningagents such as those set forth above, and flavouring agents may be addedto provide palatable oral preparations.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the antioxidant-enrichedcompositions in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those described above.Additional excipients, for example, sweetening, flavouring and colouringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oil phase may be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example liquid paraffin ormixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example, gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monoleate, and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monoleate. The emulsions may also contain-sweetening andflavouring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative and flavouringand colouring agents. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulation according to methods known in the artusing suitable dispersing or wetting agents and suspending agents suchas those mentioned above. The sterile injectable preparation may also besterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables,

Bacterial Compositions and Kits

Compositions comprising a bacterium of the invention are also provided.Examples of such bacterial compositions include, but are not limited to,starter cultures of the bacterium comprising, for example, a suitablenumber of bacteria in a small volume of growth or maintenance medium,compositions comprising the bacterium in the presence of a suitablestabiliser or carrier, compositions comprising the lyophilised bacteriumin the presence of a suitable stabiliser carrier and compositionscomprising a stab or slant of the bacteria and a suitable agar support.For probiotic use, the compositions may comprise the bacterium dispersedin a suitable foodstuff, capsule or tablet.

The present invention also provides for kits comprising a bacterium ofthe invention. The bacterium provided in the kit can be lyophilised andthe kit can additionally contain a suitable medium for reconstitution ofthe lyophilised bacterium. Alternatively the bacterium may be providedas a starter culture or a stab. The kit may further comprise suitableingredients for growth media to propagate the bacterium and/or for thefermentation of the bacterium to produce antioxidants. Individualcomponents of the kit would be packaged in separate containers and,associated with such containers, can be instructions for use.

EXAMPLES Example 1 Characterisation of Bacterium Accession No. 160103

Bacterial cultures and media. The new bacterium was isolated from thesurface of lowbush blueberries by inoculation of Tryptic Soy Broth(Difco Laboratories, Detroit Mich.), grown at 25° C. for 36 hrs, andselected on Tryptic Soy agar. MRS and Potato Dextrose agar (BectonDickinson and Company, Cockeysville Md.) were also used to determine theproperties of the bacteria. Stock cultures were maintained at −70° C. inbroth supplemented with 30% (v/v) glycerol.

Bacterial identification. Carbohydrate fermentation patterns weredetermined using API 50CH galleries as specified by the manufacturer(Bio Mérieux S A, Marcy-l'Étoile, France). API 20Strep and OF Medium(Bio Mérieux S A, Marcy-l'Étoile, France) were also used for bacterialcharacterization.

DNA methodology. The partial sequence of the 16S rRNA gene (1500 nts)was determined by MIDI Laboratories (MIDI Labs, Newark Del.) by standardprocedures, and a phylogenetic analysis based on this partial 16S rRNAfragment was performed. The sequence data were compared to sequences inthe Microseq® microbial analysis software and database (PE AppliedBiosystems).

The 16S rRNA gene was PCR amplified from genomic DNA isolated fromcolonies of the novel bacterium. The primers used for the amplificationcorrespond to positions 5, 338, 357, 515, 531, 776, 1087, 1104, 1174,1193, and 1540 in the E. coli 16S rRNA gene. Amplification products werepurified from excess primers and dNTPs using Microcon 100 (Amicon)molecular weight cut-off membranes and checked for quality and quantityby running a portion of the products on an agarose gel.

Cycle sequencing of the 16S rRNA amplification products was carried outusing AmpliTaq FS DNA polymerase and dRhodamine dye terminators. Excessdye-labeled terminators were removed from the sequencing reactions usinga Sephadex G-50 spin column. The products were collected bycentrifugation, dried under vacuum and frozen at −20° C. until ready toload. Samples were resuspended in a solution of formamide/bluedextran/EDTA and denatured prior to loading. The samples wereelectrophoresed on an ABI Prism 377 DNA Sequencer. Data was analysedusing PE Applied Biosystems DNA editing and assembly software.

Bacterial characteristics. The new bacterium isolated from lowbushblueberries was a Grain negative, catalase positive, facultativelyanaerobic coccobacillus.

From the results obtained from API 50CH, the bacteria fermentedD-glucose, D-fructose, D-mannose, arbutin, esculin, salicin, saccharoseand D-raffinose. However, the results obtained from API 20Strep showedthat the bacteria could ferment mannitol, lactose and treholase undersome conditions. The bacteria also showed positive results for VP, HIP,PYRA, α Gal, β Gal, PAL and LAP activity. Results from OF Mediumindicated that the novel bacterium has a fermentative metabolism.Results from the biochemical characterization and parameters ofincubation for the novel bacterium are compiled in Table 1. TABLE 1Biochemical characterization of novel bacterium Characteristic or testReaction^(a) Growth on: TSA at: 10° C. + 15° C. + 25° C. + (optimum) 35°C. + 37° C. − PDA + MRS − Simmons Citrate Agar + Voges-Proskauer test +OF glucose + Catalase + (strong) Acidification in the presence of:Glycerol − Erythritol − D-arabinose − L-arabinose − Ribose − D-xylose −L-xylose − Adonitol − β-methyl-D-xyloside − Galactose − D-glucose +D-fructose + D-mannose + L-sorbose − Rhamnose − Dulcitol − Inositol −Mannitol + Sorbitol − α-methyl-D-mannoside − α-methyl-D-glucoside −N-acetyl-glucosamine − Amygdalin − Arbutin − Esculin + (strong)Salicin + Cellobiose − Maltose − Lactose + Melibiose − Sucrose +Trehalose + Inulin − Melezitose − D-Raffinose + Starch − Gycogen −Xylitol − β-gentiobiose − D-turanose − D-lyxose − D-tagatose − D-fucose− L-fucose − D-arabitol − L-arabitol − Gluconate − 2-keto-gluconate −5-keto-gluconate − Enzyme production: β-glucosidase + pyrrolidonylarylamidase + α-galactosidase + β-glucuronidase − β-galactosidase +alkaline phosphatase + leucine arylamidase + arginine dihydrolase −Hydrolysis of hippurate +^(a)+, positive; −, negative

The biochemical profile and 16S rRNA gene sequence of the novelbacterium indicate that it comes from the family of facultativelyanaerobic gram-negative rods, Enterobacteriaceae. Bacteria from thisfamily have both a respiratory and fermentative type of metabolism andare catalase positive and oxidase negative.

Sequence of the 16S rRNA gene sequence for the bacterium is shown inFIG. 1 (SEQ ID NO:1), and was compared to sequences in the Microseq™microbial analysis software and database. The top ten alignment matchesare presented in a percent genetic distance format. A low percentindicates a close match. Also provided for the bacterium are neighbourjoining phylogenetic trees (Saitou and Nei, 1987, Mol. Biol. Evol., 4,406-425), generated using the top ten alignment matches (FIG. 2).

Genetic relationships are expressed in the form of Percent GeneticDifferences (% GD). A species level match may be assigned if the % GDbetween the unknown and the closest match is less than the approximateaverage % GD between species within that particular genetic family,which is usually 1% (Palys et al., 1997, IJSB, 47, 1145-1156), but canbe up to 3% (see, Vandamme, et al., (1996) Microbiol. Reviews 60:407-48;Kolbert & Persing, (1999) Curr. Microbiol, 2:299-305). A genus levelmatch will be assigned when the sequence does not meet the requirementsfor a species level match, but still clusters within the branching of awell-defined genus. For the novel bacterium, its closest match wasSerratia proteamaculans quinovora, the % GD determined was 1.82%, whichwas more than the average % GD of 1.28% between species within thefamily of Enterobacteriaceae. The % GD for other bacterial species wereas follows: Serratia grimesii 1.85% Serratia plymuthica 1.95% Hafniaalvei 1.95% Serratia proteamaculans proteamaculans 2.05% Ewingellaamericana 2.47% Serratia ficaria 2.47% Serratia fonticola 2.51% Serratiaentomophila 2.73% Yersinia frederiksenii 2.83%

The positive result obtained in the Simmons Citrate test and thefermentation of raffinose and sucrose by the bacterium indicates that itis not from the genus Hafnia (Holt and al., 1994, supra). Moreover, thenovel bacterium presents only 94% identity with Hafnia alvei, comparedto 97% of identity with Serratia proteamaculans quinovora, when the 16SrRNA gene partial sequence are analysed by BLAST 2. However, thebacterium does not ferment maltose and ribose, which is fermented by allstrains of Serratia. In addition, comparison of novel bacterium 16S rRNApartial sequence with GenBank indicates that a Rahnella genospecies isthe closest match with 98% identity to the 16S rRNA partial sequence.However, the biochemical profile of novel bacterium does not match theone of Rahnella, which demonstrates acid production from L-arabinose,cellobiose, maltose, melibiose, D-sorbitol and D-Xylose (Holt and Krieg,1984, Bergey's. Manual of Systematic Bacteriology, Vol. 1, Williams andWilkins Co., Baltimore, pp. 1-964).

Example 2 Increase in Antioxidant Capacity of Blueberries DuringFermentation with the Bacterium (Accession No. 160103)

Chemicals. Gallic acid was purchased from Acros (New Jersey, USA).Quercetin, rutin, chlorogenic acid, p-coumaric acid and sinapic acidwere purchased from Sigma-Aldrich Canada Ltd. (Ontario, Canada).

Growth of bacteria. Studied bacteria were grown in Tryptic Soy Broth(Difco Laboratories) for 24 hrs, at 22° C. Samples of the microflora ofblueberries were also plated on MRS Agar (BDH) in order to characterizethe microorganisms contained therein. Stock cultures were maintained at−70° C. in Tryptic Soy Broth supplemented with 30% (v/v) glycerol.

Preparation of blueberry mixture. Wild blueberries harvested fromdifferent areas of the Atlantic region were mixed equally to reduce thepossibility of local variations in the fruit microflora. The mixture wasprepared by blending the blueberries in a Braun Type 4259 food processorwith an equivalent volume (1:1 v/v ratio) of Minimal Broth Davis withoutdextrose (Difco laboratories). The blueberry mixture was thencentrifuged at 1700 RPM for 6 min in an IEC Centra MP4R centrifuge(international Equipment Company) to remove non homogenized particles.The resulting blueberry juice was sterilized by filtration under vacuumthrough a 0.22 μm Express Millipore filter apparatus (Millipore). Theblueberry juice was then inoculated with a saturated culture of thenovel bacterium. The quantity used was 1% of the total blueberry juicevolume, For each flask that was inoculated, a control flask was preparedunder the same conditions but without inoculation. The control contained1% of total blueberry juice volume of sterilized Tryptic Soy Broth(Difco Laboratories) in place of the bacterial culture.

Fermentation. The blueberry media were incubated in a Lab-Line lowtemperature bench top incubated shaker (Lab-Line Instruments, Inc.) at22° C. and 120 RPM for up to 7 days. Fermentations were first made withthe normal flora of blueberry by inoculation with 1% of saturated TSBculture obtained from inoculation by whole blueberries. Then,fermentations were made with the novel bacterium isolated from TSA. Acontrolled fermentation without inoculation was made in a 2.5 LiterBIOFLO 3000 fermentor (New Brunswick Scientific) by simulating pHvariation with addition of acetic acid 4N and NaOH 4N.

Sampling. Samples were taken at various times during the experiment andfiltered through 0.22 μm Millex-GP filters (Millipore). They were thenfrozen at −20° C. until further analysis. During each sampling, the pHlevel was measured with an AR15 Accumet pH meter (Fisher Scientific) andthe total soluble solids were measured with an Atago Model N1 handrefractometer. A microbial count was performed for each sample onTryptic Soy Agar Difco Laboratories) using sterilized peptone water asdilution medium. The colonies were numbered after 48 hrs of incubationat 22° C.

Phenolic compounds. The samples were analyzed using the Folin-Ciocalteumethod for measurement of total phenolics. Gallic acid was used as acalibration standard. The prepared samples were read by a μQuantMicroplate Reader (Bioteck instrument Inc.) set at a wavelength of 700nm.

Determination of Radical Scavenging Activity. Antioxidant capacity wastested using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) as a stable radical.DPPH in methanol 80% (150 μM, 200 μL) was added to 22 μL of the testcompounds at different concentrations (0-500 μM) in methanol. Eachmixture was then shaken and incubated at room temperature and in thedark. The decrease in absorbance of DPPH was measured at 30, 180 and 360minutes of incubation at 520 nm in a μQuant Microplate Reader (BioteckInstrument Inc.). Methanol was used as a blank solution. DPPH solution(200 μL) in ethanol (22 μL) served as the control. All tests wereperformed in triplicate.

A plot of A_(520nm) versus concentration of sample in the final solutionwas made for each time interval. Using the results from the timeinterval with the steepest slope, the initial slope of the curve wascalculated by linear regression (r²>0.800). The antiradical activity wasdefined by the initial slope value in units of A_(520nm)/μM of sample orμM of DPPH/μM of sample. The units were converted from A_(520nm) to μMof DPPH by developing a standard curve for DPPH using the microplatereader. The concentration of DPPH was initially determined from thecalibration curve equation given by Brand-Williams et al. (1995,Lebensm. Wiss. Technol., 28:25-30), where A_(515nm) was equal to 12509×concentration of DPPH in M-0.00258. The antiradical activity was foundto be equivalent to negative half of the antiradical power (ARP) aspreviously defined (Brand-Williams et al. 1995, Ibid). ARP was equal tothe reciprocal of the amount of compound required to decrease theinitial DPPH concentration by 50% in units of moles of DPPH per mole ofsample.

Separation by Capillary Electrophoresis. A capillary electrophoresissystem from Hewlett Packard, model G1600AX, was used for comparison ofphenolic profiles between non-fermented and fermented blueberry samples.A 64.5 cm total length, 56.0 cm effective length and 50 μm i.d. bubblefactor 3 PVA coated capillary with optical path length of 150 μm(Agilent Technologies, Inc.) was used for the separation. Samples wereinjected using 50.0 mbar pressure, for 30.0 seconds. The separation wasmade using borate 50 mM pH 9.25 as buffer, under maintained temperatureof 25° C., and voltage of 25 kV set at positive polarity, and current to120 μA. Detection of compounds was made using diode array detection at280, 345, 520 and 200 nm.

Quantification of the phenolic acid compounds by HPLC. The method usedto separate the phenolic compounds on HPLC was derived from Kalt and al.(1999, Can. J. Plant Sci., 79:617-623). A 50 μL injection of sample wasseparated on a Zorbax SB-C18 Rapid Resolution 4.6 mm i.d.×150 mm, 3.5 μmassociated to a ODS-Hypersil (C-18) guard column 2.1 mm i.d.×20 mm, 5 μmfrom Agilent Technologies Inc. using a Agilent HPLC 1100 series systemequipped with a quaternary pump and a Diode Array Detector. Data werecollected at 270 nm, 520 nm, 340 nm, 320 nm, and 365 nm, with 800 nm setas reference. Compounds were separated with 5% formic acid in water(solvent B) and methanol (solvent D), using a gradient elution program;0-10.24 min, 14-17% D; 10.24-35.28 min, 17-23% D; 35.28-64.59 min,23-47% D; 64.59-66.59 min, 47-14% D; 66.59-70 min, 14% D. Flow rate was1.0 mL min⁻¹.

Column temperature was maintained at 30° C. during separation. Sampleswere filtered through a 0.22 μm Millex-GP filter unit (Millipore,Canada) prior to injection. HPLC quantification was made by comparisonwith gallic acid as reference for comparison between peak heights.

Statistical Analysis. Data: represent the mean of three replicateanalyses tested. Results were processed for statistical significanceusing Student's t test. Differences at P<0.05 were considered to besignificant.

Fermentation with fruit surface microflora. An increase in totalphenolic content from fermentation was obtained with the fruit normalflora of wild blueberries (Table 2). Fermentation conditions were 22°C., 120 RPM and an aerobic environment. The decrease in pH of the mediumand an increase in bacterial plate counts on TSA were in concordancewith the increase in total phenolics during fermentation. Total solublesolids, as indicated by the °Brix, decreased during fermentation. On day3, numerous bacteria were isolated from TSA plate counts to find onebacterium that would be responsible for the increase in phenoliccontent. These bacteria were then tested in fermentation to determinetheir effect on the total phenolic content of wild blueberry samples.TABLE 2 Results obtained during fermentation of wild blueberry by thenormal microflora found on fruit surface. Time (days) Bacterial counts°Brix pH Total phenolics^(a) 0 4 6.1 ± 0.2 4.74 ± 0.03 166.84 ± 22.86 12.0 × 10² 6.1 ± 0.1 4.47 ± 0.01  99.36 ± 10.70 3 3.5 × 10⁸ 6.0 ± 0.13.66 ± 0.04 176.57 ± 6.65  5 2.8 × 10⁷ 4.0 ± 0.1 3.32 ± 0.04 409.32 ±84.52 7 ND 3.1 ± 0.1 3.35 ± 0.07 372.46 ± 45.11^(a)Total phenolics are expressed in mg of gallic acid equivalent/100 gof fresh weight.ND indicates not determined.

Fermentation with the bacterium. Fermentation was conducted in thepresence of the bacterium at 22° C., 120 RPM under aerobic conditions.To prevent phenolic degradation by heat, filtration through 0.22 μmfilters was used as a method of sterilizing the juice. A controlfermentation was conducted under the same conditions, but using aninoculum of sterile TSB. Negative controls were run under the sameconditions. A noticeable increase in the total phenolic content of wildblueberry juice occurred after only 1 day of incubation, indicating thatthe bacterium plays an important role in the increase of total phenoliccontent (Table. 3). Total soluble solids, as demonstrated by the °Brix,decreased from day one (6.40±0.05) to day seven (3.87±0.11) offermentation. An interesting-phenomenon during fermentation was PHvariation. The pH changed from day one (4.73±0.01) to reach the lowestpoint on day 3 (3.31±0.05), followed by an increase on day 7(5.09±0.17). The increase in pH was concomitant with a decrease inbacterial count. No significant variation in total phenolics, pH and°Brix was observed for the controlled fermentation.

A control fermentation conducted using standard Medium in place of theberry juice medium did not result in any increase in phenolicantioxidant content of the medium.

A fermentation simulation was conducted to study the effect of pHvariation on phenolic content. This fermentation involved a sterilizedblueberry medium in which the pH was adjusted over time by addition ofacetic acid and sodium hydroxide to simulate the pH variation thatoccurred during fermentation with the novel bacterium. No significantvariation in total phenolic content was observed during the 6 days thatthe fermentation simulation was conducted (see, FIG. 3 and Table 3).TABLE 3 Total phenolic content during fermentation of wild blueberry bythe bacterium. Total Phenolic Content (mg of gallic Day of acidequivalent/100 g of fresh weight) Fermentation Control Fermentation pHSimulation 0 168.25 ± 3.55  158.12 ± 4.88  105.27 ± 1.99  1 125.13 ±27.87 364.03 ± 20.11 98.98 ± 4.48 2 154.19 ± 11.91 374.66 ± 14.64 90.71± 2.69 3 150.75 ± 3.77  347.94 ± 12.00 102.63 ± 12.19 4 132.16 ± 16.36369.03 ± 14.47 98.53 ± 0.93 5 115.59 ± 26.91 410.59 ± 33.44 99.36 ± 2.947 147.00 ± 4.46  392.63 ± 19.43 101.44 ± 3.89 

Radical Scavenging Activity. Samples collected from the blueberryfermentation with the bacterium were assessed for radical scavengingactivity (RSA) using the DPPH method (FIG. 4). Such analysis has beenpreviously associated with the study of antioxidant capacity(Brand-Williams, W., et al., (1995) Lebensm. Wiss. Technol., 28, 25-30).Increase in RSA was not only associated with an increase in totalphenolic content during fermentation, but also with a change in thephenolic profile, resulting in the production of phenolic compounds withbetter antioxidant capacity, as shown in FIGS. 4A and 4B. However,during the first three days of fermentation, the increase in RSA for day1 and 2 was followed by a decrease for day 4 (FIGS. 4C and 4D). Thisincrease in RSA was not only associated with the increase in totalphenolic content, but also with a change in the phenolic profile offermented blueberry. In contrast, the loss in RSA on day 3, of thefermentation can be attributed to an alteration of the phenolic profile,but not to a loss in total phenolics (FIGS. 4C and 4D). Another way topresent the RSA, is by the antiradical activity and antiradical power,indicative of the antioxidant capacity (Tables 4 and 5). These resultswere calculated from results obtained from the DPPH method. TABLE 4Antiradical activity of fermented blueberry samples for DPPH radical(total phenolic concentrations being adjusted to the initial value ofthe samples) Blueberry sample Antiradical activity^(a) Antiradicalpower^(b) Non fermented after 3 days −1.985 ± 0.349 3.970 After 3 daysof fermentation −3.367 ± 0.237 6.733 Day 0 of fermentation −2.904 ±0.124 5.809 Day 1 of fermentation −4.437 ± 0.062 8.874 Day 2 offermentation −4.599 ± 0.022 9.197 Day 3 of fermentation −4.176 ± 0.1838.352^(a)Values are means of slope coefficients calculated by linearregression ± standard deviations (n = 3) in μM of DPPH/μM of blueberrysample in GAE.^(b)Antiradical power was defined as the reciprocal of the amount ofantioxidant needed to decrease the initial DPPH concentration by 50%.The antiradical activity was equivalent to the negative half of theantiradical power.

TABLE 5 Total phenolic concentrations not adjusted to the initial valueof samples Blueberry sample Antiradical activity^(a) Antiradicalpower^(b) Non fermented after 3 days −2.021 ± 0.269 4.042 After 3 daysof fermentation −4.857 ± 0.060 9.714 Day 0 of fermentation −2.076 ±0.352 4.152 Day 1 of fermentation −4.954 ± 0.049 9.909 Day 2 offermentation −4.849 ± 0.071 9.699 Day 3 of fermentation −4.606 ± 0.1099.211^(a)Values are means of slope coefficients calculated by linearregression ± standard deviations (n = 3) in μM of DPPH/μM of blueberrysample in GAE.^(b)Antiradical power was defined as the reciprocal of the amount ofantioxidant needed to decrease the initial DPPH concentration by 50%.The antiradical activity was equivalent to the negative half of theantiradical power.

Sample separation by capillary electrophoresis. Phenolic profiles forblueberry fermented with the bacterium and the control fermentation weredetermined by capillary electrophoresis (EC) in order to identify anymajor changes (FIG. 5A). From the EC results, the increase in totalphenolics during fermentation could be attributed to the production of aparticular phenolic compound, as shown by the presence of a main peak onthe phenolic profile of fermented blueberry. UV spectra of the mainpeak, as shown in FIG. 5B, is characterized by a strong absorbance at200 nm, followed by a plateau from 215 nm to 235 nm, and a second strongabsorbance at 280 nm. The UV spectra for this compound confirmed that itwas not a flavonoid structure, such as an anthocyanin or a flavonol,because of the absence of absorbance peaks in the 520 nm and 350 nmregions, respectively, and, therefore, is likely to be of the phenolicacid type.

Quantification and characterization of phenolic acid compounds by HPLC.HPLC was also used to characterize the phenolic acid compounds obtainedfrom fermented blueberry sample with bacterium. Quercetin could not bedetected from phenolic profiles of non-fermented and fermented blueberrysamples by HPLC spiking. However, rutin could be detected at anapproximate concentration of (1.22±0.02) mg/100 g FW in non-fermentedblueberry samples after 3 days of incubation. Significant decrease inrutin content to (1.09±0.03) mg/100 g FW could be observed in fermentedblueberry samples with the same incubation time (P<0.05). Production ofgallic acid has been observed during blueberry fermentation, as shown byHPLC spiking. Gallic acid went from a non-detectable concentration onday 0 to a concentration of (6.46±0.05) mg/100 g FW on day 3 ofblueberry fermentation peak 1, FIG. 5D). However, gallic acidconcentration varied from one fermentation to the other, and could be aslow as (2.67±0.09) mg/100 g FW after 3 days of fermentation. Increase ingallic acid content could be attributed to hydrolysable tannindegradation during the fermentation process. Content of chlorogenic acidafter 3 days of blueberry fermentation was (85.27±0.28) mg/100 g FW. Nosignificant difference could be observed in the chlorogenic acidconcentration between fermented and unfermented blueberry (P<0.05) (peak3, FIGS. 5C and D). No p-coumaric acid and sinapic acid were detected inany of the fermented and unfermented blueberry samples. Peak 2 (FIG. 5D)was produced only after fermentation of blueberry with the bacterium ata concentration of (64.20±0.13) mg of gallic acid equivalents/100 g FWafter 3 days of incubation. No significant variation was observedbetween day 1 and day 5 of fermentation for concentration of the newcompound (P<0.05).

From results obtained in his example, it was concluded that increase inantioxidant capacity during fermentation was not only attributed toincrease in total phenolics, but also to a change in the phenolicprofile as shown by HPLC.

Overall, it was found that the bacterium isolated from the normalsurface micro-flora of blueberry is mainly responsible for the increasein total phenolics observed during fermentation by the normalmicro-flora found on the fruit surface. Fermentation by this bacteriumalso increases the antioxidant capacity of blueberries, as demonstratedby the radical scavenging activity, not only from increase in totalphenolics, but also from a change in the phenolic profile, asdemonstrated by the production of gallic acid and other compounds.

Example 3 Increase in Antioxidant Capacity of Cranberries DuringFermentation with the Bacterium (Accession No. 160103)

Preparation of cranberry mixture. Cranberries were purchased fromCanneberges Acadiennes (Richibouctou Village, NB) and the resultingcranberry mixture was produced utilizing the method described in thepreceding example, with minor modifications. The quantity of saturatedculture of the bacterium utilized to inoculate the juice was 2% of thetotal juice volume. For each flask that was inoculated, a control flaskwas prepared under the same conditions, but using 2% of the total juicevolume of sterilized Tryptic Soy Broth in place of the bacterialinoculum.

Fermentation The cranberry juice media was fermented with the bacteriumas described in example 2, for up to 4 days.

Sampling and Analysis of total phenolics, radical scavenging activityand HPLC were performed as described in the preceding example.

Fermentation with the bacterium. Fermentation of cranberry juice wasconducted in the presence of the bacterium under aerobic conditions andresulted in a noticeable increase in the total phenolic content of thefermented cranberry juice after only 1 day of incubation, indicatingthat the bacterium plays an important role in the increase of totalphenolic content. (FIG. 6A). An interesting phenomenon duringfermentation was pH variation (FIG. 7). Cranberry juice pH decreasedfrom 4.13 to the minimum value 3.56 after 2 days of fermentation andincreased to 6.54 by the 10^(th) day of fermentation. The mixture colourchanged simultaneously with the pH, from dark red at the beginning tolight rose, dark green, brown and black at the end. This pH variationaffected the phenolic content of fermented cranberry juice.

Fermentation with the bacterium under anaerobic conditions Similarfermentation was conducted under anaerobic conditions in a 2.5 LiterBIOFLO 3000 fermentor (New Brunswick Scientific). The phenolic contentof fermented cranberry juice increased under anaerobic conditions (FIG.8A).

Fermentation with the novel bacterium and wine yeast (Saccharomycescerevisiae). Fermentation of cranberry juice was conducted in thepresence of the bacterium and wine yeast under anaerobic conditions. Thephenolic content of the fermented cranberry juice also increased underthese conditions (FIG. 8B). This experiment demonstrated thecompatibility of the bacterium with Saccharomyces cerevisiae.

Radical scavenging activity. Samples collected from the cranberry juicefermentation with the bacterium were studied for radical scavengingactivity (RSA), using the DPPH method as described in the precedingexample. Another way to present the RSA is by the antiradical activityand antiradical power, indicative of the antioxidant capacity (FIG. 9).These results were calculated from data obtained by the DPPH method.Table 6 shows antiradical activity as translated from the DPPH method.TABLE 6 The antiradical activity and antiradical power of cranberryjuice Extract Antiradical activity Antiradical power Unfermentedcranberry juice Day 0 −1.15 (0.02) 2.30 (0.06) Day 1 −1.17 (0.03) 2.34(0.06) Day 2 −1.18 (0.02) 2.37 (0.04) Day 3 −1.16 (0)   2.33 (0)   Day 4−1.14 (0.02) 2.29 (0.04) Day 8 −1.16 (0.04) 2.31 (0.09) Day 9 −1.14(0.05) 2.29 (0.11) Day 10 −1.15 (0.03) 2.30 (0.06) Fermented cranberryjuice (pH not controlled) Day 0 −1.12 (0)   2.25 (0)   Day 1 −1.82(0.02) 3.65 (0.05) Day 2 −3.43 (0.20) 6.86 (0.39) Day 3 −3.47 (0.09)6.94 (0.19) Day 4 −3.41 (0.15) 6.82 (0.29) Day 8 −3.91 (0.07) 7.82(0.14) Day 9 −4.02 (0.04) 8.04 (0.08) Day 10 −3.95 (0.08) 7.89 (0.16)Fermented cranberry juice (controlled pH) Day 0 −1.01 (0.03) 2.01 (0.06)Day 1 −1.09 (0.02) 2.18 (0.05) Day 2 −1.22 (0.03) 2.44 (0.06) Day 3−1.33 (0.05) 2.65 (0.11) Day 4 −2.15 (0.02) 4.30 (0.05) Day 8 −3.90(0.05) 7.89 (0.11) Day 9 −4.58 (0.06) 9.16 (0.13) Day 10 −4.78 (0.16)9.56 (0.31) Gallic acid* −6.21 (0.60) 12.50 Chlorogenic acid* −5.08(0.29) 10.16*Fukumoto and Mazza (2000)

Quantification and characterization of phenolic acid compounds by HPLC.HPLC was used to analyze the changes in phenolic profiles of fermentedcranberry juice as previously described with blueberry juice. Quercetin,Rutin, Chlorogenic acid, Sinapic acid, and p-coumaric acid were used todetermine their content in non fermented and fermented cranberry juicesby HPLC spiking. Unlike blueberry juice, none of these compounds weredetected with the HPLC method used (data not shown). Production ofgallic acid was observed during cranberry fermentation, as shown by HPLCspiking.

Example 4 Effects of Fermented Cranberry and Blueberry Juices on theInhibition of Nitric Oxide Production

Preparation of blueberry and cranberry mixture. The blueberry andcranberry mixtures were produced utilizing the methods described in thepreceding examples. Briefly, the quantity of saturated culture of thebacterium utilized: to inoculate the juice was 2% of the total juicevolume. For each flask that was inoculated, a control flask was preparedunder the same conditions, but without inoculation. Instead, a quantityof 2% of the total juice volume of sterilized Tryptic Soy Broth wasadded to the mixture.

Fermentation The blueberry and cranberry juice media were fermented withthe novel bacterium as described in the preceding examples, for up to 4days.

Cell culture. The mouse monocyte/macrophage cell line RAW 264.7 gammaNO(−) [American Type Culture Collection (ATCC)] was cultured inRPMI-1640 supplemented with 10% (v/v) heat-inactivated PBS, streptomycin(100 mg/ml) and penicillin (100 units/ml). All cultures were incubatedat 37° C. in a humidified atmosphere with 5% CO₂. Cell number wasassessed by trypan blue dye exclusion on a Neubauer hemacytometer. Cellswere grown to 90% confluence in sterile cell culture flasks and gentlydetached using a scraper (Fisher Scientific). For phenolic compoundtreatment tests, cells were cultured in triplicate in Costar flat-bottomcell culture plates (Corning Inc.). Cells were plated at a density of6×10⁵ cells/well in 24-well cell culture plates and grown for 1 h toallow them to attach to the plate. Compounds to be tested were initiallydissolved in 10 μl of DMSO, and then RPMI was added to make solutions ina series of concentrations with a dilution factor of 2. The finalconcentrations of test compound that cells received were 16, 31, 63,125, 250, and 500 μM, respectively. For berry juices, the finalconcentrations were 16, 31, 63, 125, 250 and 500 μM GAE, respectively.Cells were supplemented with the test compounds for 1 h beforestimulation with 10 ng/ml lipopolysaccharide (LPS) and 10-50 units/mlinterferon gamma (IFN-γ). The activated cells were incubated for 24 hand then supernatants were collected to determine nitrite concentrationand/or stored at −80° C. for further use. Control cells were grown underidentical conditions but were not exposed to the test compounds orLPS/IFN-γ.

Nitric Oxide (NO) Determination. Nitrite concentration was used as anindication of NO production. The procedure for NO determination wasbased on the Griess reaction. The assay was assessed as in the methodsof Wang and Mazza (2002a, 2002b) for cell supernatants supplemented withphenolic compounds or berry juices. In brief, 100 μL of cell culturesupernatant or sodium nitrite standard was mixed with an equal volume ofGriess reagent [a mixture of 0.1% (w/v) N-(1-naphthyl)-ethylenediaminedihydrochloride and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric, acid,the two parts being mixed together within 1 h of use] using a 96-wellplate. A set of parallel analyses was also conducted by applying only100 μL of 2.5% (v/v) phosphoric acid instead of Griess reagent to eachof 100 μL of cell supernatants or the mixture of standard sodium nitriteand cyanidin chloride. After 20 min at room temperature, the absorbanceat 540 nm was measured using the microtitration reader. The netabsorbance of the product of the Griess reaction was obtained bysubtracting that of anthocyanins from the total. The absorbance wasreferred to a nitrite standard curve to determine the nitriteconcentration in the supernatants.

Cell Viability. Cell viability was determined by the MTT assay and/orthe resazurin-based in vitro toxicology assay kit, TOX-8 (Sigma). Theviability of cells activated only by LPS/IFN-γ was arbitrarily set as100, and all other viabilities, that is, cells receiving differenttreatments but within the same 24-well plate, were normalized to that ofthe LPS/IFN-γ activated control cells.

Tumour necrosis factor alpha (TNF-α) Quantification. TNF-α in cellsupernatants was determined by using an OptELISA set mouse TNF-α(mono/poly) from BD PharMingen. The enzyme-linked immunosorbent assay(ELISA) was carried out as specified by the manufacturer.

Statistical Analysis. Data represent the mean of three replicateanalyses tested. Results were processed for statistical significanceusing Student's t test. Differences at P<0.05 were considered to besignificant.

Inhibition of Nitric Oxide Production. RAW 264.7 gamma NO(−) cell linewas derived from the RAW 264.7 mouse monocyte/macrophage cell line.Unlike the parental line, RAW 264.7 gamma NO(−) does not produce nitricoxide upon treatment with IFN-γ alone, but requires LPS for fullactivation. When LPS/IFN-γ was administered to RAW 264.7 gamma (NO-)macrophages, NO production, measured as nitrite, increased dramaticallyfrom the basal level of 0 to ≧30 μM after 24 h. To determine the effectsof cranberry and blueberry juice and other phenolic compounds on NOproduction, different concentrations of these compounds (500, 250, 125,63, and 16 μM) were incubated with the LPS/IFN-γ-activated macrophages(Table 7). Cell viability was assayed to exclude the possibility thatthe inhibitory effects of phenolic compounds were due to theircytotoxicity. The MTT assay was used for the colorless phenoliccompound-treated macrophages, whereas the resazurin assay was used forcranberry and blueberry juice-treated macrophages.

The inhibitory effects (IE) was expressed as the percentage decrease ofNO production asIE (%)=100−[NO] ^(a) [NO] ^(b)×100Where [NO]^(a) represents the NO concentration in supernatants from bothphenolic compounds supplemented and LPS/IFN-γ-activated macrophages and[NO]^(b) represents the NO concentration in supernatants fromLPS/IFN-γ-activated control macrophages.

Gallic acid, Chlorogenic Acid. The inhibitory effect of gallic acid onNO production in LPS/IFN-γ-activated macrophages was dose-dependent(Table 7). The NO production decreased with the increase inconcentration of gallic acid in the media. For example, the 250 and 500μM gallic acid treatments attained 23 and 30% reduction in NOproduction, respectively without cytotoxicity toward the cells.Chlorogenic acid, although a very strong antioxidant in vitro (Table 6),showed no inhibitory effects on RAW 264.7 gamma (NO-) macrophages;instead, it induced production of NO (Table 7).

Cranberry and blueberry juice. Cranberry juice contains high levels ofphenolic compounds (500 mg GAE/L). This concentration was increaseddramatically after fermentation with the bacteria (≧3000 mg GAE/L). Itwas expected that fermented juice would demonstrate inhibitory activityon NO production in LPS/IFN-γ-activated macrophages. The inhibition onNO production by berry juices is shown in Table 7 and FIGS. 10 and 11.The unfermented blueberry juice exhibited a small effect (3.5%) on NOproduction but only at 500 μM concentration. Using fermented juice, asimilar inhibitory effect was attained at 125 μM concentration. At highconcentration (250 μM), blueberry juice exhibited a strong inhibition onNO production (40%) with slight toxicity on the macrophages. On theother hand, the unfermented cranberry juice had a significant effect onNO production (22%) at 125 μM without toxicity. The crude fermentedcranberry juice attained an inhibition of 61% only at 31 μM. Higherconcentrations of crude fermented cranberry juice had more inhibitoryeffects but they also increased the cytotoxicity on macrophages. Thefermented cranberry juice after being adjusted showed less cytotoxicitywhile it still had a significant inhibitory effect 65% at 250 μM. TABLE7 Inhibition of macrophage NO production by berry juices and phenoliccompounds Concen- Inhibitory tration Cell viability effect Extract (μM)Nitrite (μM) (%) (%) Cranberry juice Control 0  100 (0.4) (unfermented)LPS/IFN-γ 30.6 (0.6)  100 (0.8) 16 29.6 (0.9) 100.1 (2.8)   5.1 (2.8) 3127.5 (0.5) 99.4 (6.5)   10 (1.5) 63 25.6 (0.5) 98.2 (2.7) 16.3 (1.8) 12523.7 (1.2) 102.5 (3.2)  22.2 (4)   250 16.7 (0.7) 76.4 (7.5) 45.4 (2.3)500 0 18.9 (5.8) Cranberry juice Control 0  100 (1.2) (fermented andLPS/IFN-γ 30.8 (0.6)  100 (0.8) adjusted) 16 32.8 (0.8) 101.6 (1.2)  6.6 (1.8) 31 30.6 (0.8) 98.7 (1.4)  6.5 (2.5) 63 30.1 (0.3) 98.2 (1.7) 8.1 (0.9) 125 24.9 (1.5) 95.1 (1.5)   24 (4.6) 250 11.4 (0.4) 92.9(1.8)   65 (1.2) 500 0 68.8 (6.5) Cranberry juice Control 0  100 (0.3)(fermented) LPS/IFN-γ 30.6 (0.6)  100 (0.8) 16 23.8 (0.9) 93.9 (3.6)22.2 (3.1) 31 11.8 (0.4) 95.8 (3.6) 61.3 (1.2) 63  0.9 (0.3) 86.3 (2.1)97.0 (1)   125 0 21.8 (3.6) 250 0   1 (1.2) 500 0 0 Blueberry juiceControl 0  100 (0.4) (unfermented) LPS/IFN-γ 35.2 (0.6)  100 (0.7) 1635.3 (0.2) 103.3 (1)   0 31 37.0 (0.4) 104.1 (0.9)  0 63 35.4 (1.2)100.8 (2.6)  0 125 37.7 (0.4) 102.3 (2.8)  0 250 37.5 (1.7)   97 (0.6) 0500 33.9 (1.1) 99.8 (2.9)  3.5 (3.1) Blueberry juice Control 0  100(1.1) (fermented and LPS/IFN-γ 36.3 (1.8)  100 (0.3) 0 adjusted) 16 40.1(1.5) 99.6 (0.8) 0 31 41.3 (0.6) 99.3 (3.2) 0 63 37.7 (0.8) 98.9 (3.3) 0125 33.9 (0.8) 99.3 (3.5)  4.6 (1.5) 250 21.7 (0.3) 93.8 (1.3) 39.9(0.4) 500  0.3 (0.5) 52.6 (2.3) 99.3 (1.3) Gallic acid Control 0  100(2.1) LPS/IFN-γ 32.7 (2)    100 (7.7) 16 36.2 (3.7)  110 (2.2) 31 35.4(2.9)  111 (6.4) 63 29.3 (0.3) 98.9 (0.9) 10.5 (0.9) 125 31.1 (5.9)108.5 (0.6)    19 (4.3) 250 25.2 (1.5) 100.6 (3.7)    23 (4.5) 500 22.6(0.6) 109.7 (5.4)  30.9 (1.9) Chlorogenic Control 0  100 (3.2) acidLPS/IFN-γ 33.8 (1.8)  100 (4.6) 16 37.1 (0.4) 102.4 (6.7)  −9.6 (1.3) 3139 (1) 105.6 (8.7)  −15.2 (2.9)  63  40.4 (1.64)  106 (5.7) −19.5 (4.9) 125 41.7 (1.7) 98.5 (7.1) −23.4 (5)   250 47.6 (1.4) 102.3 (6.9)  −40.8(4.2)  500 46.6 (1.5) 102.8 (4.2)  −37.9 (4.5) 

TNF-α Quantification. Gallic acid and Chlorogenic acid may induce TNF-αproduction in LPS/IFN-γ-activated macrophages but not in a concentrationdependant manner. This may explain the fermented berry juiced inducedproduction of macrophage TNF-α, as the fermentation process with thebacterium produced gallic acid. Furthermore, chlorogenic acidrepresented a significant proportion of the blueberry juice phenoliccontent. However, only unfermented blueberry juice showed an inductiveeffect in a concentration-dependant manner, whereas unfermentedcranberry juice or fermented blueberry and cranberry juice increaseddramatically the production of TNF-α in LPS/IFN-γ-activated RAW 264.7gamma NO(−) macrophages. The effect of gallic acid, chlorogenic acid,unfermented and fermented berry juices is shown Table 8. TABLE 8Induction of macrophage TNF-α production Extract Concentration (μM)TNF-α (%) Cell viability (%) Cranberry juice Control  2.9 (1.5) 105.7(1.8)  (unfermented) LPS/IFN-γ  100 (0.7)  100 (1.3) 50  184 (5.8) 101.1(1.8)  100 179.5 (4.4)  104.2 (0.9)  200 109.9 (7.4)  92.8 (1.4) 30073.02 (5)   55.1 (4.9) 400 22.6 (2.8) 49.6 (6.0) 500 15.3 (3.6) 47.3(6.4) Cranberry juice Control  2.7 (2.7) 105.7 (1.8)  (fermented andLPS/IFN-γ  100 (3.6)  100 (1.3) adjusted) 50 359.1 (6.7)  101.8 (3.2) 100 431.4 (19.3) 106.3 (1.2)  200 465.8 (21.8) 102.5 (4.1)  300 456.2(20.5) 99.8 (1.7) 400 448.8 (13.8) 93.04 (3.2)  500 311.2 (13.8) 88.1(2.1) Blueberry juice Control  6.1 (3.7)  100 (0.4) (unfermented)LPS/IFN-γ  100 (0.6) 100 (2)  50 368.6 (8)   98.8 (0.7) 100 432.8 (8.8) 95.6 (2.2) 200 491.5 (36.4)  100 (2.1) 300 502.7 (37.4) 98.7 (0.6) 400518.1 (20.9) 99.1 (1.8) 500 666.9 (27.7) 98.5 (1.8) Blueberry juiceControl  5.6 (3.6)  100 (1.1) (fermented and LPS/IFN-γ  100 (7.5)  100(2.2) adjusted) 50 779.7 (25.5) 99.1 (0.9) 100 940.9 (33.8)   97 (2.9)200 1026.5 (9.8)  100.7 (1.4)  300 1122.1 (8.3)  97.7 (3.5) 400 1112.4(4.9)  100.5 (1.1)  500 907.9 (6.2)  98.8 (1.7) Gallic acid Control 7.6(7)  97.4 (5.1) LPS/IFN-γ  100 (3.5)  100 (0.5) 50 103.4 (3.2)  97.7(0.4) 100 126.2 (11)   98.3 (2.3) 200 160.3 (8.4)  96.7 (0.8) 300 257.4(36.7) 97.6 (2.5) 400 213.9 (54.8) 96.1 (2.3) 500 183.5 (19.8) 95.6(1.6) Chlorogenic Control  6.5 (3.3)  100 (3.2) acid LPS/IFN-γ   100(15.3)  100 (2.2) 50 105.7 (8.4)   102 (0.4) 100 101.5 (5.4)  100.6(1.9)  200 99.2 (7.6) 98.9 (2.3) 300 102.3 (15.6) 97.1 (1.4) 400   126(31.1) 97.3 (1)   500 115.6 (15.4)   99 (1.9)

Example 5 Fermentation with Grapes

The ability of the bacterium (Accession No. 160103) to ferment a mediumcontaining juice from green seedless grapes was assessed as described,above for blueberries and cranberries. The grapes used were President'schoice Organics™ green seedless grapes, purchased from Loblaws Inc.,Montreal. The results, as shown in Table 9 and FIG. 6B, indicate thatfermentation with the bacterium can increase the total phenolic contentof a medium comprising green grape juice. Red and black organic grapescan also be tested as described above for green grapes. TABLE 9 Phenoliccontent during fermentation of green grapes by the bacterium. PhenolicConcentration Extract Time/day (mg GAE/L) Unfermented green grapes 0133.7 (2.9) 1 126.7 (3.4) 2 128.6 (7.5) 3 123.2 (1.2) 4 128.1 (1.2) 5131.9 (4.6) Fermented green grapes 0 135.3 (2.8) 1 172.1 (6.5) 2  297.3(16.2) 3 401.0 (4.2) 4 442.3 (2.3) 5  512.6 (20.2)

Example 6 Fermentation with Other Berries

Preliminary results have also indicated that the bacterium (AccessionNo. 160103) can be successfully fermented with media prepared using thefollowing berries:

Elderberries (Samucus canadensis)

Chokecherry (Prunus virginiana)

Blackcurrant (Ribes americanum and/or Ribes nigrum).

The bacterium will also be tested for fermentation with Saskatoonberries. A mixture of Saskatoon berries from four different varietieswill be used: Thiessen, Honeywood, Northline and Smokey varieties. Theberries are hand-picked, cleaned of leaves, twigs, etc. and frozenwithin 24 hours of harvest. All the berries are from the Summer, 2003crop.

Example 7 In Vivo Testing of the Antioxidant-Enriched Fermented FruitExtracts

In order to investigate further the immunomodulatory effects of thefermented fruit extracts, the following in vivo tests can be conducted.

The effect of the fermented Suit extracts on tumoral and mucosalimmunity can be investigated in BALB/c mice using protocols as describedby Matar, C., et al., (J. Dairy Res. (2001) 68(4): 601-609). Mice willbe fed the fermented fruit extract under investigation orally at days,2, 5 and 7. The effect of antioxidants on the pathways leading toactivation of NF-kB is well known. This effect can be measured byfollowing the parameters of tumour growth over a 3 month period usingstandard techniques. The parameters to be followed are: 1) the rate oftumour development; 2) histopathological studies (haematoxin eosinstain); 3) apoptosis (Tunel method); 4) phagocytic index (PI) ofperitoneal macrophages; 5) presence of specific cytokines for the NF-kBpathway, together with TNF-α, within the infiltrative tumour cells(mammalian or fibrosarcomas). In addition, the effect of the fermentedfruit extract on the Gut Associated Lymphoid Tissues can be studied bymeasuring the ex vivo peritoneal macrophage activities in order todetermine the effect on the pro-inflammatory cytokines profile.

The in vivo biological activity of the fermented fruit extracts can alsobe tested using animal models of diabetes or insulin resistance. Invitro bio-assays of insulin secretion and action can be used tocomplement these studies.

The in vivo biological activity of the fermented fruit extracts againstfree-radical and oxidative stress in the central nervous system can betested in an appropriate animal model in order to assess the ability ofthe extracts to ameliorate neurodegenerative diseases, such asAlzheimer's, or conditions associated therewith.

The disclosure of all patents, publications, including published patentapplications, and database entries referenced in this specification arespecifically incorporated by reference in their entirety to the sameextent as if each such individual patent, publication, and databaseentry were specifically and individually indicated to be incorporated byreference.

The embodiments of the invention being thus described, it will beobvious that the same may be varied in many ways. Such variations arenot to be regarded as a departure from the spirit and scope of theinvention, and all such modifications as would be obvious to one skilledin the art are intended to be included within the scope of the followingclaims.

1. A bacterial strain having all the identifying characteristics of thebacterium deposited under Accession Number
 160103. 2. A bacterial strainhaving a 16S rRNA gene comprising a nucleotide sequence that is at leastabout 97% identical to the sequence as set forth in SEQ ID NO:1.
 3. Thebacterial strain according to claim 2, wherein said 16S rRNA genecomprises a nucleotide sequence that is at least about 98% identical tothe sequence as set forth in SEQ ID NO:1.
 4. The bacterial strainaccording to claim 2, wherein said 16S rRNA gene comprises a nucleotidesequence that is at least about 98.2% identical to the sequence as setforth in SEQ ID NO:1.
 5. The bacterial strain according to any one ofclaims 2 to 4, wherein said bacterial strain is capable of fermentingfruit extracts to increase the antioxidant content thereof.
 6. A processfor producing an antioxidant-enriched fruit extract comprising:fermenting a fruit extract with the bacterial strain according to anyone of claims 1 to
 5. 7. The process according to claim 6, wherein saidfruit extract is a berry extract.
 8. The process according to claim 7,wherein said berry extract is a blueberry, cranberry, chokecherry,elderberry, blackcurrant, or Saskatoon berry extract, or a combinationthereof.
 9. The process according to claim 7, wherein said berry extractis derived from berries of the genus Vaccinium.
 10. The processaccording to claim 6, wherein said fruit extract is a grape extract. 11.An antioxidant-enriched fruit extract produced by the process accordingto any one of claims 6 to
 10. 12. An antioxidant composition comprisinga carrier or diluent and an antioxidant-enriched fruit extract, saidfruit extract produced by the process according to any one of claims 6to
 10. 13. A process for producing an antioxidant-enriched fruitextract, said method comprising: (a) providing a sterile mediumcomprising a fruit extract; (b) inoculating said sterile medium with abacterial strain having all the identifying characteristics of thebacterium deposited under Accession Number 160103 to provide a bacterialculture; (c) fermenting said bacterial culture to provide anantioxidant-enriched fruit extract, and (d) recovering saidantioxidant-enriched fruit extract.
 14. The process according to claim13, wherein said fruit extract is a berry extract.
 15. The processaccording to claim 14, wherein said berry extract is a blueberry,cranberry, chokecherry, elderberry, blackcurrant, or Saskatoon berryextract, or a combination thereof.
 16. The process according to claim14, wherein said berry extract is derived from berries of the genusVaccinium.
 17. The process according to claim 13, wherein said fruitextract is a grape extract.
 18. The process according to any one ofclaims 13 to 17, wherein said bacterial culture is fermented at atemperature between about 8° C. and about 36° C.
 19. The processaccording to any one of claims 13 to 18, wherein the starting pH of saidmedium is above 3.2.
 20. The process according to any one of claims 13to 19, wherein said bacterial culture is fermented under aerobicconditions.
 21. The process according to any one of claims 13 to 19,wherein said bacterial culture is fermented under anaerobic conditions.22. An antioxidant-enriched fruit extract produced by the processaccording to any one of claims 13 to
 21. 23. An antioxidant compositioncomprising a carrier or diluent and an antioxidant-enriched fruitextract, said fruit extract produced by the process according to any oneof claims 13 to
 21. 24. Use of the antioxidant-enriched fruit extractaccording to claim 11 or 22 in the preparation of a cosmeticcomposition.
 25. Use of the antioxidant-enriched fruit extract accordingto claim 11 or 22 in the preparation of a pharmaceutical composition.26. Use of the antioxidant-enriched Suit extract according to claim 11or 22 in the preparation of a nutraceutical, functional food, beverageor dietary supplement.
 27. Use of the antioxidant-enriched fruit extractaccording to claim 11 or 22 to deliver antioxidants to a mammal in needthereof.
 28. A cosmetic composition produced by a process comprising thesteps of: (a) fermenting a fruit extract with the bacterial strainaccording to any one of claims 1 to 5 to provide an antioxidant-enrichedfruit extract; and (b) combining said antioxidant-enriched fruit extractwith a cosmetically acceptable carrier or diluent.
 29. A pharmaceuticalcomposition produced by a process comprising the steps of: (a)fermenting a fruit extract with the bacterial strain according to anyone of claims 1 to 5 to provide an antioxidant-enriched fruit extract;and (b) combining said antioxidant-enriched fruit extract with apharmaceutically acceptable carrier or diluent.
 30. A cosmeticcomposition comprising the antioxidant-enriched fruit extract accordingto claim 11 or 22 and a cosmetically acceptable carrier or diluent. 31.A pharmaceutical composition comprising the antioxidant-enriched fruitextract according to claim 11 or 22 and a pharmaceutically acceptablecarrier or diluent.
 32. Use of the pharmaceutical composition accordingto claim 29 or 31 as an antioxidant in a mammal in need thereof.
 33. Useof the pharmaceutical composition according to claim 29 or 31 as animmunomodulator in a mammal in need thereof.
 34. Use of thepharmaceutical composition according to claim 29 or 31 to stimulateTNF-α production in a mammal in need thereof.
 35. Use of the bacterialstrain according to any one of claims 1 to 5 in a fermentation process.36. Use of the bacterial strain according to any one of claims 1 to 5 asa food additive.
 37. Use of the bacterial strain according to any one ofclaims 1 to 5 to increase the antioxidant content of a food product. 38.Use of a bacterial strain according to any one of claims 1 to 5 tometabolise toxic phenolic compounds in a sample.
 39. A compositioncomprising the bacterial strain according to any one of claims 1 to 5and a suitable medium or stabiliser.
 40. A kit comprising the bacterialstrain according to any one of claims 1 to 5 and optionally one or moregrowth medium ingredient for propagation of the bacterial strain.